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Sample records for polyubiquitin linkage-specific antibodies

  1. Linkage-specific conformational ensembles of non-canonical polyubiquitin chains

    PubMed Central

    Castañeda, Carlos A.; Chaturvedi, Apurva; Camara, Christina M.; Curtis, Joseph E.; Krueger, Susan; Fushman, David

    2015-01-01

    Polyubiquitination is a critical protein post-translational modification involved in a variety of processes in eukaryotic cells. The molecular basis for selective recognition of the polyubiquitin signals by cellular receptors is determined by the conformations polyubiquitin chains adopt; this has been demonstrated for K48- and K63-linked chains. Recent studies of the so-called non-canonical chains (linked via K6, K11, K27, K29, or K33) suggest they play important regulatory roles in growth, development, and immune system pathways, but biophysical studies are needed to elucidate the physical/structural basis of their interactions with receptors. A first step towards this goal is characterization of the conformations these chains adopt in solution. We assembled diubiquitins (Ub2) comprised of every lysine linkage. Using solution NMR measurements, small-angle neutron scattering (SANS), and in silico ensemble generation, we determined population-weighted conformational ensembles that shed light on the structure and dynamics of the non-canonical polyubiquitin chains. We found that polyubiquitin is conformationally heterogeneous, and each chain type exhibits unique conformational ensembles. For example, K6-Ub2 and K11-Ub2 (at physiological salt concentration) are in dynamic equilibrium between at least two conformers, where one exhibits a unique Ub/Ub interface, distinct from that observed in K48-Ub2 but similar to crystal structures of these chains. Conformers for K29-Ub2 and K33-Ub2 resemble recent crystal structures in the ligand-bound state. Remarkably, a number of diubiquitins adopt conformers similar to K48-Ub2 or K63-Ub2, suggesting potential overlap of biological function among different lysine linkages. These studies highlight the potential power of determining function from elucidation of conformational states. PMID:26422168

  2. Polyubiquitination of Insulin-like Growth Factor I Receptor (IGF-IR) Activation Loop Promotes Antibody-induced Receptor Internalization and Down-regulation*

    PubMed Central

    Mao, Yifan; Shang, Yonglei; Pham, Victoria C.; Ernst, James A.; Lill, Jennie R.; Scales, Suzie J.; Zha, Jiping

    2011-01-01

    Ubiquitination has been implicated in negatively regulating insulin-like growth factor I receptor (IGF-IR) activity. Because of the relative stability of IGF-IR in the presence of ligand stimulation, IGF-IR ubiquitination sites have yet to be mapped and characterized, thus preventing a direct demonstration of how the receptor ubiquitination contributes to downstream molecular cascades. We took advantage of an anti-IGF-IR antibody (h10H5) that induces more efficient receptor down-regulation to show that IGF-IR is promptly and robustly ubiquitinated. The ubiquitination sites were mapped to the two lysine residues in the IGF-IR activation loop (Lys-1138 and Lys-1141) and consisted of polyubiquitin chains formed through both Lys-48 and Lys-29 linkages. Mutation of these ubiquitinated lysine residues resulted in decreased h10H5-induced IGF-IR internalization and down-regulation as well as a reduced cellular response to h10H5 treatment. We have therefore demonstrated that IGF-IR ubiquitination contributes critically to the down-regulating and antiproliferative activity of h10H5. This finding is physiologically relevant because insulin-like growth factor I appears to mediate ubiquitination of the same major sites as h10H5 (albeit to a lesser extent), and ubiquitination is facilitated by pre-existing phosphorylation of the receptor in both cases. Furthermore, identification of a breast cancer cell line with a defect in IGF-IR ubiquitination suggests that this could be an important tumor resistance mechanism to evade down-regulation-mediated negative regulation of IGF-IR activity in cancer. PMID:21994939

  3. K63 polyubiquitination is a new modulator of the oxidative stress response

    PubMed Central

    Silva, Gustavo M.; Finley, Daniel; Vogel, Christine

    2014-01-01

    Ubiquitination is a post-translational modification that signals multiple processes, including protein degradation, trafficking, and DNA repair. Polyubiquitin accumulates globally during the oxidative stress response, which has been mainly attributed to increased ubiquitin conjugation and perturbations in protein degradation. Here we show that the unconventional K63-linked polyubiquitin accumulates in the yeast Saccharomyces cerevisiae subjected to peroxides in a highly sensitive and regulated manner. We demonstrated that hydrogen peroxide inhibits the deubiquitinating enzyme Ubp2 leading to accumulation of K63 conjugates assembled by the Rad6-Bre1 ubiquitin conjugase and ligase, respectively. Using linkage-specific isolation methods and SILAC-based quantitative proteomics, we identified >100 new K63 polyubiquitinated targets, which were significantly enriched in ribosomal proteins. Finally, we demonstrated that impairment of K63 ubiquitination during oxidative stress impacts polysome stability and protein expression, rendering cells more sensitive to stress, revealing a new redox-regulatory role for this modification. PMID:25622294

  4. New conformations of linear polyubiquitin chains from crystallographic and solution-scattering studies expand the conformational space of polyubiquitin.

    PubMed

    Thach, Trung Thanh; Shin, Donghyuk; Han, Seungsu; Lee, Sangho

    2016-04-01

    The conformational flexibility of linkage-specific polyubiquitin chains enables ubiquitylated proteins and their receptors to be involved in a variety of cellular processes. Linear or Met1-linked polyubiquitin chains, associated with nondegradational cellular signalling pathways, have been known to adopt multiple conformations from compact to extended conformations. However, the extent of such conformational flexibility remains open. Here, the crystal structure of linear Ub2 was determined in a more compact conformation than that of the previously known structure (PDB entry 3axc). The two structures differ significantly from each other, as shown by an r.m.s.d. between C(α) atoms of 3.1 Å. The compactness of the linear Ub2 structure in comparison with PDB entry 3axc is supported by smaller values of the radius of gyration (Rg; 18 versus 18.9 Å) and the maximum interatomic distance (Dmax; 55.5 versus 57.8 Å). Extra intramolecular hydrogen bonds formed among polar residues between the distal and proximal ubiquitin moieties seem to contribute to stabilization of the compact conformation of linear Ub2. An ensemble of three semi-extended and extended conformations of linear Ub2 was also observed by small-angle X-ray scattering (SAXS) analysis in solution. In addition, the conformational heterogeneity in linear polyubiquitin chains is clearly manifested by SAXS analyses of linear Ub3 and Ub4: at least three distinct solution conformations are observed in each chain, with the linear Ub3 conformations being compact. The results expand the extent of conformational space of linear polyubiquitin chains and suggest that changes in the conformational ensemble may be pivotal in mediating multiple signalling pathways.

  5. Structural Basis for Specific Recognition and Cleavage of Polyubiquitin Chains

    NASA Astrophysics Data System (ADS)

    Fukai, Shuya; Sato, Yusuke

    Ubiquitin is a conserved 76-residue protein that is reversibly conjugated with substrate proteins to regulate various cellular processes. The terminal carboxyl group of ubiquitin can be bonded to lysine residues of substrate proteins including ubiquitin itself. All seven lysine residues (K6, K11, K27, K29, K33, K48 and K63) and the N-terminal Met can act as receptors for the conjugation, producing polyubiquitin chains. For instance, the most abundant K48-linked chains serve as signals for proteasomal degradation, whereas K63-linked chains function in other proteasome-independent processes. Here we show how these functionally and structurally distinct chains are discriminated by linkage-specific deubiquitinating enzymes and binding proteins.

  6. Polyubiquitin binding to ABIN1 is required to prevent autoimmunity

    PubMed Central

    Venigalla, Ram K.C.; Ordureau, Alban; Patterson-Kane, Janet C.; Powell, David W.; Toth, Rachel; C. Arthur, J. Simon

    2011-01-01

    The protein ABIN1 possesses a polyubiquitin-binding domain homologous to that present in nuclear factor κB (NF-κB) essential modulator (NEMO), a component of the inhibitor of NF-κB (IκB) kinase (IKK) complex. To address the physiological significance of polyubiquitin binding, we generated knockin mice expressing the ABIN1[D485N] mutant instead of the wild-type (WT) protein. These mice developed all the hallmarks of autoimmunity, including spontaneous formation of germinal centers, isotype switching, and production of autoreactive antibodies. Autoimmunity was suppressed by crossing to MyD88−/− mice, demonstrating that toll-like receptor (TLR)–MyD88 signaling pathways are needed for the phenotype to develop. The B cells and myeloid cells of the ABIN1[D485N] mice showed enhanced activation of the protein kinases TAK, IKK-α/β, c-Jun N-terminal kinases, and p38α mitogen-activated protein kinase and produced more IL-6 and IL-12 than WT. The mutant B cells also proliferated more rapidly in response to TLR ligands. Our results indicate that the interaction of ABIN1 with polyubiquitin is required to limit the activation of TLR–MyD88 pathways and prevent autoimmunity. PMID:21606507

  7. OTU Deubiquitinases Reveal Mechanisms of Linkage Specificity and Enable Ubiquitin Chain Restriction Analysis

    PubMed Central

    Mevissen, Tycho E.T.; Hospenthal, Manuela K.; Geurink, Paul P.; Elliott, Paul R.; Akutsu, Masato; Arnaudo, Nadia; Ekkebus, Reggy; Kulathu, Yogesh; Wauer, Tobias; El Oualid, Farid; Freund, Stefan M.V.; Ovaa, Huib; Komander, David

    2013-01-01

    Summary Sixteen ovarian tumor (OTU) family deubiquitinases (DUBs) exist in humans, and most members regulate cell-signaling cascades. Several OTU DUBs were reported to be ubiquitin (Ub) chain linkage specific, but comprehensive analyses are missing, and the underlying mechanisms of linkage specificity are unclear. Using Ub chains of all eight linkage types, we reveal that most human OTU enzymes are linkage specific, preferring one, two, or a defined subset of linkage types, including unstudied atypical Ub chains. Biochemical analysis and five crystal structures of OTU DUBs with or without Ub substrates reveal four mechanisms of linkage specificity. Additional Ub-binding domains, the ubiquitinated sequence in the substrate, and defined S1’ and S2 Ub-binding sites on the OTU domain enable OTU DUBs to distinguish linkage types. We introduce Ub chain restriction analysis, in which OTU DUBs are used as restriction enzymes to reveal linkage type and the relative abundance of Ub chains on substrates. PMID:23827681

  8. Structure-guided reprogramming of human cGAS dinucleotide linkage specificity.

    PubMed

    Kranzusch, Philip J; Lee, Amy S Y; Wilson, Stephen C; Solovykh, Mikhail S; Vance, Russell E; Berger, James M; Doudna, Jennifer A

    2014-08-28

    Cyclic dinucleotides (CDNs) play central roles in bacterial pathogenesis and innate immunity. The mammalian enzyme cGAS synthesizes a unique cyclic dinucleotide (cGAMP) containing a 2'-5' phosphodiester linkage essential for optimal immune stimulation, but the molecular basis for linkage specificity is unknown. Here, we show that the Vibrio cholerae pathogenicity factor DncV is a prokaryotic cGAS-like enzyme whose activity provides a mechanistic rationale for the unique ability of cGAS to produce 2'-5' cGAMP. Three high-resolution crystal structures show that DncV and human cGAS generate CDNs in sequential reactions that proceed in opposing directions. We explain 2' and 3' linkage specificity and test this model by reprogramming the human cGAS active site to produce 3'-5' cGAMP, leading to selective stimulation of alternative STING adaptor alleles in cells. These results demonstrate mechanistic homology between bacterial signaling and mammalian innate immunity and explain how active site configuration controls linkage chemistry for pathway-specific signaling.

  9. The HLA-DRα Chain Is Modified by Polyubiquitination*

    PubMed Central

    Lapaque, Nicolas; Jahnke, Martin; Trowsdale, John; Kelly, Adrian P.

    2009-01-01

    Ubiquitination plays a major role in regulating cell surface and intracellular localization of major histocompatibility complex class II molecules. Two E3 ligases, MARCH I and MARCH VIII, have been shown to polyubiquitinate lysine residue 225 in the cytoplasmic tail of I-Aβ and HLA-DRβ. We show that lysine residue 219 in the cytoplasmic tail of DRα is also subject to polyubiquitination. Each chain of the HLA-DR heterodimer is independently recognized and ubiquitinated, but DRβ is more extensively modified. In the cytoplasmic tail of DRβ lysine, residue 225 is the only residue that is absolutely required for ubiquitination; all other residues can be deleted or substituted without loss of function. In contrast, although lysine 219 is absolutely required for modification of DRα, other features of the DRα tail act to limit the extent of ubiquitination. PMID:19117940

  10. Monoubiquitination joins polyubiquitination as an esteemed proteasomal targeting signal.

    PubMed

    Livneh, Ido; Kravtsova-Ivantsiv, Yelena; Braten, Ori; Kwon, Yong Tae; Ciechanover, Aaron

    2017-06-01

    A polyubiquitin chain attached covalently to the target substrate has been recognized for long as the "canonical" proteasomal degradation signal. However, several proteins have been shown to be targeted for degradation following monoubiquitination, indicating that the proteasome can recognize signals other than a ubiquitin polymer. A comprehensive screen aiming at determining the extent of this mode of recognition revealed that ∼40% of mammalian and ∼20% of yeast proteins are degraded following monoubiquitination. Characterization of these proteins showed that on average, the monoubiquitinated proteins are smaller than the polyubiquitinated ones, and in humans, are less disordered. Further, proteins degraded by the two different modes belong to distinct functional groups. These findings along with detailed structural analysis of the proteasome, its ubiquitin receptors and deubiquitinating enzymes, suggest that the ubiquitin signal - its formation, recognition, editing, and removal - is far more complex and diverse than originally assumed. Also see the video abstract here: https://youtu.be/QKpN9c6Rg20. © 2017 WILEY Periodicals, Inc.

  11. Antibody

    MedlinePlus

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...

  12. Tissue and serum alpha 2-3- and alpha 2-6-linkage specific sialylation changes in oral carcinogenesis.

    PubMed

    Shah, Manisha H; Telang, Shaila D; Shah, Pankaj M; Patel, Prabhudas S

    2008-04-01

    Increased sialylation of cell surface glycoconjugates is among the key molecular changes associated with malignant transformation and cancer progression. We investigated significance of linkage-specific sialylation changes in oral carcinogenesis. Tissue and serum levels of total sialic acid (TSA), linkage-specific sialyltransferases (ST) and sialoproteins were analyzed from patients with oral precancerous conditions (OPC) and oral cancer as well as the post-treatment follow-up blood samples of oral cancer patients. TSA levels were measured using a spectrophotometric method. The linkage-specific lectins, Sambusus nigra (SNA) and Maackia amurensis (MAM) detects alpha 2-6- and alpha 2-3-linked sialic acid, respectively, were used to analyze ST activity and sialoproteins. Malignant tissues showed significantly higher levels of TSA, reactivity of SNA and MAM, and alpha 2,3-ST activity compared to the adjacent normal tissues. alpha 2,6-ST was also higher in malignant tissues. Similarly, the marker levels were higher in precancerous tissues than their adjacent normal tissues. Serum levels of TSA, TSA/ total proteins, alpha 2-6-sialoproteins and alpha 2,6-ST were markedly increased in untreated oral cancer patients compared to the controls and OPC as well as responder (CR) patients. Serum levels of the markers were higher or comparable between untreated oral cancer patients and non-responders (NR). Serum levels of alpha 2-3-sialylation were elevated in non-responders compared with the responders. Further, the observed sialylation changes in tissue and serum were found to be associated with various clinicopathological features and disease progression. Thus, the data suggest potential utility of sialylation markers in early detection, prognostication and treatment monitoring of oral cancer.

  13. Non-hydrolyzable Diubiquitin Probes Reveal Linkage-Specific Reactivity of Deubiquitylating Enzymes Mediated by S2 Pockets

    PubMed Central

    Flierman, Dennis; van der Heden van Noort, Gerbrand J.; Ekkebus, Reggy; Geurink, Paul P.; Mevissen, Tycho E.T.; Hospenthal, Manuela K.; Komander, David; Ovaa, Huib

    2016-01-01

    Summary Ubiquitin chains are important post-translational modifications that control a large number of cellular processes. Chains can be formed via different linkages, which determines the type of signal they convey. Deubiquitylating enzymes (DUBs) regulate ubiquitylation status by trimming or removing chains from attached proteins. DUBs can contain several ubiquitin-binding pockets, which confer specificity toward differently linked chains. Most tools for monitoring DUB specificity target binding pockets on opposing sides of the active site; however, some DUBs contain additional pockets. Therefore, reagents targeting additional pockets are essential to fully understand linkage specificity. We report the development of active site-directed probes and fluorogenic substrates, based on non-hydrolyzable diubiquitin, that are equipped with a C-terminal warhead or a fluorogenic activity reporter moiety. We demonstrate that various DUBs in lysates display differential reactivity toward differently linked diubiquitin probes, as exemplified by the proteasome-associated DUB USP14. In addition, OTUD2 and OTUD3 show remarkable linkage-specific reactivity with our diubiquitin-based reagents. PMID:27066941

  14. Synergistic effect of two E2 ubiquitin conjugating enzymes in SCFhFBH1 catalyzed polyubiquitination

    PubMed Central

    Choi, Jin Sun; Kim, Sunhong; Kim, Kidae; Myung, Pyung Keun; Park, Sung Goo; Seo, Yeon-Soo; Park, Byoung Chul

    2015-01-01

    Ubiquitination is a post translational modification which mostly links with proteasome dependent protein degradation. This process has been known to play pivotal roles in the number of biological events including apoptosis, cell signaling, transcription and translation. Although the process of ubiquitination has been studied extensively, the mechanism of polyubiquitination by multi protein E3 ubiquitin ligase, SCF complex remains elusive. In the present study, we identified UbcH5a as a novel stimulating factor for poly-ubiquitination catalyzed by SCFhFBH1 using biochemical fractionations and MALDI-TOF. Moreover, we showed that recombinant UbcH5a and Cdc34 synergistically stimulate SCFhFBH1 catalyzed polyubiquitination in vitro. These data may provide an important cue to understand the mechanism how the SCF complex efficiently polyubiquitinates target substrates. [BMB Reports 2015; 48(1): 25-29] PMID:24667174

  15. Molecular basis for specificity of the Met1-linked polyubiquitin signal.

    PubMed

    Elliott, Paul R

    2016-12-15

    The post-translational modification of proteins provides a rapid and versatile system for regulating all signalling pathways. Protein ubiquitination is one such type of post-translational modification involved in controlling numerous cellular processes. The unique ability of ubiquitin to form polyubiquitin chains creates a highly complex code responsible for different subsequent signalling outcomes. Specialised enzymes ('writers') generate the ubiquitin code, whereas other enzymes ('erasers') disassemble it. Importantly, the ubiquitin code is deciphered by different ubiquitin-binding proteins ('readers') functioning to elicit particular cellular responses. Ten years ago, the methionine1 (Met1)-linked (linear) polyubiquitin code was first identified and the intervening years have witnessed a seismic shift in our understanding of Met1-linked polyubiquitin in cellular processes, particularly inflammatory signalling. This review will discuss the molecular mechanisms of specificity determination within Met1-linked polyubiquitin signalling.

  16. Molecular basis for specificity of the Met1-linked polyubiquitin signal

    PubMed Central

    Elliott, Paul R.

    2016-01-01

    The post-translational modification of proteins provides a rapid and versatile system for regulating all signalling pathways. Protein ubiquitination is one such type of post-translational modification involved in controlling numerous cellular processes. The unique ability of ubiquitin to form polyubiquitin chains creates a highly complex code responsible for different subsequent signalling outcomes. Specialised enzymes (‘writers’) generate the ubiquitin code, whereas other enzymes (‘erasers’) disassemble it. Importantly, the ubiquitin code is deciphered by different ubiquitin-binding proteins (‘readers’) functioning to elicit particular cellular responses. Ten years ago, the methionine1 (Met1)-linked (linear) polyubiquitin code was first identified and the intervening years have witnessed a seismic shift in our understanding of Met1-linked polyubiquitin in cellular processes, particularly inflammatory signalling. This review will discuss the molecular mechanisms of specificity determination within Met1-linked polyubiquitin signalling. PMID:27913667

  17. Polyubiquitin insertions and the phylogeny of Cercozoa and Rhizaria.

    PubMed

    Bass, David; Moreira, David; López-García, Purificación; Polet, Stephane; Chao, Ema E; von der Heyden, Sophie; Pawlowski, Jan; Cavalier-Smith, Thomas

    2005-08-01

    A single or double amino acid insertion at the monomer-monomer junction of the universal eukaryotic protein polyubiquitin is unique to Cercozoa and Foraminifera, closely related 'core' phyla in the protozoan infrakingdom Rhizaria. We screened 11 other candidate rhizarians for this insertion: Radiozoa (polycystine and acantharean radiolaria), a 'microheliozoan', and Apusozoa; all lack it, supporting suggestions that Foraminifera are more closely related to Cercozoa than either is to other eukaryotes. The insertion's size was ascertained for 12 additional Cercozoa to help resolve their basal branching order. The earliest branching Cercozoa generally have a single amino acid insertion, like all Foraminifera, but a large derived clade consisting of all Monadofilosa except Metopion, Helk-esimastix, and Cercobodo agilis has two amino acids, suggesting one doubling event and no reversions to a single amino acid. Metromonas and Sainouron, cercozoans of uncertain position, have a double insertion, suggesting that they belong in Monadofilosa. An alternative interpretation, suggested by the higher positions for Metopion and Cercobodo on Bayesian trees compared with most distance trees, cannot be ruled out, i.e. that the second insertion took place earlier, in the ancestral filosan, and was followed by three independent reversions to a single amino acid in Chlorarachnea, Metopion and Cercobodo.

  18. Parkin promotes proteasomal degradation of synaptotagmin IV by accelerating polyubiquitination.

    PubMed

    Kabayama, Hiroyuki; Tokushige, Naoko; Takeuchi, Makoto; Kabayama, Miyuki; Fukuda, Mitsunori; Mikoshiba, Katsuhiko

    2017-04-01

    Parkin is an E3 ubiquitin ligase whose mutations cause autosomal recessive juvenile Parkinson's disease (PD). Unlike the human phenotype, parkin knockout (KO) mice show no apparent dopamine neuron degeneration, although they demonstrate reduced expression and activity of striatal mitochondrial proteins believed to be necessary for neuronal survival. Instead, parkin-KO mice show reduced striatal evoked dopamine release, abnormal synaptic plasticity, and non-motor symptoms, all of which appear to mimic the preclinical features of Parkinson's disease. Extensive studies have screened candidate synaptic proteins responsible for reduced evoked dopamine release, and synaptotagmin XI (Syt XI), an isoform of Syt family regulating membrane trafficking, has been identified as a substrate of parkin in humans. However, its expression level is unaltered in the striatum of parkin-KO mice. Thus, the target(s) of parkin and the molecular mechanisms underlying the impaired dopamine release in parkin-KO mice remain unknown. In this study, we focused on Syt IV because of its highly homology to Syt XI, and because they share an evolutionarily conserved lack of Ca(2+)-binding capacity; thus, Syt IV plays an inhibitory role in Ca(2+)-dependent neurotransmitter release in PC12 cells and neurons in various brain regions. We found that a proteasome inhibitor increased Syt IV protein, but not Syt XI protein, in neuron-like, differentiated PC12 cells, and that parkin interacted with and polyubiquitinated Syt IV, thereby accelerating its protein turnover. Parkin overexpression selectively degraded Syt IV protein, but not Syt I protein (indispensable for Ca(2+)-dependent exocytosis), thus enhancing depolarization-dependent exocytosis. Furthermore, in parkin-KO mice, the level of striatal Syt IV protein was increased. Our data indicate a crucial role for parkin in the proteasomal degradation of Syt IV, and provide a potential mechanism of parkin-regulated, evoked neurotransmitter release

  19. Mechanism of Polyubiquitination by Human Anaphase-Promoting Complex: RING Repurposing for Ubiquitin Chain Assembly

    DOE PAGES

    Brown, Nicholas G.; Watson, Edmond R.; Weissmann, Florian; ...

    2014-10-09

    Polyubiquitination by E2 and E3 enzymes is a predominant mechanism regulating protein function. Some RING E3s, including anaphase-promoting complex/cyclosome (APC), catalyze polyubiquitination by sequential reactions with two different E2s. An initiating E2 ligates ubiquitin to an E3-bound substrate. Another E2 grows a polyubiquitin chain on the ubiquitin-primed substrate through poorly defined mechanisms. Here in this paper we show that human APC’s RING domain is repurposed for dual functions in polyubiquitination. The canonical RING surface activates an initiating E2-ubiquitin intermediate for substrate modification. However, APC engages and activates its specialized ubiquitin chain-elongating E2 UBE2S in ways that differ from current paradigms.more » During chain assembly, a distinct APC11 RING surface helps deliver a substrate-linked ubiquitin to accept another ubiquitin from UBE2S. Our data define mechanisms of APC/UBE2S-mediated polyubiquitination, reveal diverse functions of RING E3s and E2s, and provide a framework for understanding distinctive RING E3 features specifying ubiquitin chain elongation.« less

  20. Mechanism of polyubiquitination by human Anaphase Promoting Complex: RING repurposing for ubiquitin chain assembly

    PubMed Central

    Brown, Nicholas G.; Watson, Edmond R.; Weissmann, Florian; Jarvis, Marc A.; VanderLinden, Ryan; Grace, Christy R. R.; Frye, Jeremiah J.; Qiao, Renping; Dube, Prakash; Petzold, Georg; Cho, Shein Ei; Alsharif, Omar; Bao, Ju; Davidson, Iain F.; Zheng, Jie; Nourse, Amanda; Kurinov, Igor; Peters, Jan-Michael; Stark, Holger; Schulman, Brenda A.

    2014-01-01

    Polyubiquitination by E2 and E3 enzymes is a predominant mechanism regulating protein function. Some RING E3s, including Anaphase Promoting Complex/Cyclosome (APC), catalyze polyubiquitination by sequential reactions with two different E2s. An initiating E2 ligates ubiquitin to an E3-bound substrate. Another E2 grows a polyubiquitin chain on the ubiquitin-primed substrate through poorly defined mechanisms. Here we show that human APC’s RING domain is repurposed for dual functions in polyubiquitination. The canonical RING surface activates an initiating E2~ubiquitin intermediate for substrate modification. However, APC engages and activates its specialized ubiquitin chain elongating E2 UBE2S in ways that differ completely from current paradigms. During chain assembly, a distinct APC11 RING surface helps deliver a substrate-linked ubiquitin to accept another ubiquitin from UBE2S. Our data define mechanisms of APC/UBE2S-mediated polyubiquitination, reveal unexpectedly diverse functions of RING E3s and E2s, and provide a framework for understanding distinctive RING E3 features specifying ubiquitin chain elongation. PMID:25306923

  1. CYLD, a deubiquitinase specific for lysine63-linked polyubiquitins, accumulates at the postsynaptic density in an activity-dependent manner

    SciTech Connect

    Dosemeci, Ayse; Thein, Soe; Yang, Yijung; Reese, Thomas S.; Tao-Cheng, Jung-Hwa

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer CYLD is a deubiquitinase specific for lysine63-linked polyubiquitins. Black-Right-Pointing-Pointer Presence of CYLD in PSDs is established by biochemistry and immunoEM. Black-Right-Pointing-Pointer CYLD accumulates on PSDs upon depolarization of neurons. Black-Right-Pointing-Pointer Accumulation of CYLD at PSDs may regulate trafficking/degradation of synaptic proteins. -- Abstract: Polyubiquitin chains on proteins flag them for distinct fates depending on the type of polyubiquitin linkage. While lysine48-linked polyubiquitination directs proteins to proteasomal degradation, lysine63-linked polyubiquitination promotes different protein trafficking and is involved in autophagy. Here we show that postsynaptic density (PSD) fractions from adult rat brain contain deubiquitinase activity that targets both lysine48 and lysine63-linked polyubiquitins. Comparison of PSD fractions with parent subcellular fractions by Western immunoblotting reveals that CYLD, a deubiquitinase specific for lysine63-linked polyubiquitins, is highly enriched in the PSD fraction. Electron microscopic examination of hippocampal neurons in culture under basal conditions shows immunogold label for CYLD at the PSD complex in approximately one in four synapses. Following depolarization by exposure to high K+, the proportion of CYLD-labeled PSDs as well as the labeling intensity of CYLD at the PSD increased by more than eighty percent, indicating that neuronal activity promotes accumulation of CYLD at the PSD. An increase in postsynaptic CYLD following activity would promote removal of lysine63-polyubiquitins from PSD proteins and thus could regulate their trafficking and prevent their autophagic degradation.

  2. Regulation of tumor suppressor EAF2 polyubiquitination by ELL1 and SIAH2 in prostate cancer cells.

    PubMed

    Yu, Xinpei; Ai, Junkui; Cai, Liquan; Jing, Yifeng; Wang, Dan; Dong, Jun; Pascal, Laura E; Zhang, Jian; Luo, Rongcheng; Wang, Zhou

    2016-05-17

    RNA Polymerase II Elongation Factor (ELL)-associated factor 2 (EAF2) is a tumor suppressor frequently down-regulated in human prostate cancer. We previously reported that its binding partner ELL1 can enhance EAF2 protein stability and activity. Here we show that EAF2 can be polyubiquitinated and its degradation blocked by proteasome inhibitor. Co-immunoprecipitation detected EAF2 binding to SIAH2, an E3 ligase, and SIAH2 overexpression enhanced polyubiquitination of EAF2. Co-transfection of EAF2 binding partner ELL1 blocked EAF2 ubiquitination, providing a mechanism for EAF2 stabilization. Finally, EAF2K81R mutant, which exhibits reduced polyubiquitination and increased stability, was more potent than wild-type EAF2 in apoptosis induction. These findings suggest that SIAH2 is an E3 ligase for EAF2 polyubiquitination and ELL1 can enhance EAF2 level and function by blocking its polyubiquitination.

  3. Regulation of tumor suppressor EAF2 polyubiquitination by ELL1 and SIAH2 in prostate cancer cells

    PubMed Central

    Yu, Xinpei; Ai, Junkui; Cai, Liquan; Jing, Yifeng; Wang, Dan; Dong, Jun; Pascal, Laura E.; Zhang, Jian; Luo, Rongcheng; Wang, Zhou

    2016-01-01

    RNA Polymerase II Elongation Factor (ELL)-associated factor 2 (EAF2) is a tumor suppressor frequently down-regulated in human prostate cancer. We previously reported that its binding partner ELL1 can enhance EAF2 protein stability and activity. Here we show that EAF2 can be polyubiquitinated and its degradation blocked by proteasome inhibitor. Co-immunoprecipitation detected EAF2 binding to SIAH2, an E3 ligase, and SIAH2 overexpression enhanced polyubiquitination of EAF2. Co-transfection of EAF2 binding partner ELL1 blocked EAF2 ubiquitination, providing a mechanism for EAF2 stabilization. Finally, EAF2K81R mutant, which exhibits reduced polyubiquitination and increased stability, was more potent than wild-type EAF2 in apoptosis induction. These findings suggest that SIAH2 is an E3 ligase for EAF2 polyubiquitination and ELL1 can enhance EAF2 level and function by blocking its polyubiquitination. PMID:27058417

  4. Walnut diet reduces accumulation of polyubiquitinated proteins and inflammation in the brain of aged rats

    USDA-ARS?s Scientific Manuscript database

    An increase in the aggregation of misfolded/damaged polyubiquitinated proteins has been the hallmark of many age-related neurodegenerative diseases. The accumulation of these potentially toxic proteins in brain increases with age, in part due to increased oxidative and inflammatory stresses. Walnuts...

  5. Lysine 63-linked polyubiquitin chain may serve as a targeting signal for the 26S proteasome

    PubMed Central

    Saeki, Yasushi; Kudo, Tai; Sone, Takayuki; Kikuchi, Yoshiko; Yokosawa, Hideyoshi; Toh-e, Akio; Tanaka, Keiji

    2009-01-01

    Recruitment of substrates to the 26S proteasome usually requires covalent attachment of the Lys48-linked polyubiquitin chain. In contrast, modifications with the Lys63-linked polyubiquitin chain and/or monomeric ubiquitin are generally thought to function in proteasome-independent cellular processes. Nevertheless, the ubiquitin chain-type specificity for the proteasomal targeting is still poorly understood, especially in vivo. Using mass spectrometry, we found that Rsp5, a ubiquitin-ligase in budding yeast, catalyzes the formation of Lys63-linked ubiquitin chains in vitro. Interestingly, the 26S proteasome degraded well the Lys63-linked ubiquitinated substrate in vitro. To examine whether Lys63-linked ubiquitination serves in degradation in vivo, we investigated the ubiquitination of Mga2-p120, a substrate of Rsp5. The polyubiquitinated p120 contained relatively high levels of Lys63-linkages, and the Lys63-linked chains were sufficient for the proteasome-binding and subsequent p120-processing. In addition, Lys63-linked chains as well as Lys48-linked chains were detected in the 26S proteasome-bound polyubiquitinated proteins. These results raise the possibility that Lys63-linked ubiquitin chain also serves as a targeting signal for the 26S proteaseome in vivo. PMID:19153599

  6. PiZ Mouse Liver Accumulates Polyubiquitin Conjugates That Associate with Catalytically Active 26S Proteasomes

    PubMed Central

    Haddock, Christopher J.; Blomenkamp, Keith; Gautam, Madhav; James, Jared; Mielcarska, Joanna; Gogol, Edward; Teckman, Jeffrey; Skowyra, Dorota

    2014-01-01

    Accumulation of aggregation-prone human alpha 1 antitrypsin mutant Z (AT-Z) protein in PiZ mouse liver stimulates features of liver injury typical of human alpha 1 antitrypsin type ZZ deficiency, an autosomal recessive genetic disorder. Ubiquitin-mediated proteolysis by the 26S proteasome counteracts AT-Z accumulation and plays other roles that, when inhibited, could exacerbate the injury. However, it is unknown how the conditions of AT-Z mediated liver injury affect the 26S proteasome. To address this question, we developed a rapid extraction strategy that preserves polyubiquitin conjugates in the presence of catalytically active 26S proteasomes and allows their separation from deposits of insoluble AT-Z. Compared to WT, PiZ extracts had about 4-fold more polyubiquitin conjugates with no apparent change in the levels of the 26S and 20S proteasomes, and unassembled subunits. The polyubiquitin conjugates had similar affinities to ubiquitin-binding domain of Psmd4 and co-purified with similar amounts of catalytically active 26S complexes. These data show that polyubiquitin conjugates were accumulating despite normal recruitment to catalytically active 26S proteasomes that were available in excess, and suggest that a defect at the 26S proteasome other than compromised binding to polyubiquitin chain or peptidase activity played a role in the accumulation. In support of this idea, PiZ extracts were characterized by high molecular weight, reduction-sensitive forms of selected subunits, including ATPase subunits that unfold substrates and regulate access to proteolytic core. Older WT mice acquired similar alterations, implying that they result from common aspects of oxidative stress. The changes were most pronounced on unassembled subunits, but some subunits were altered even in the 26S proteasomes co-purified with polyubiquitin conjugates. Thus, AT-Z protein aggregates indirectly impair degradation of polyubiquitinated proteins at the level of the 26S proteasome

  7. The unexpected role of polyubiquitin chains in the formation of fibrillar aggregates.

    PubMed

    Morimoto, Daichi; Walinda, Erik; Fukada, Harumi; Sou, Yu-Shin; Kageyama, Shun; Hoshino, Masaru; Fujii, Takashi; Tsuchiya, Hikaru; Saeki, Yasushi; Arita, Kyohei; Ariyoshi, Mariko; Tochio, Hidehito; Iwai, Kazuhiro; Namba, Keiichi; Komatsu, Masaaki; Tanaka, Keiji; Shirakawa, Masahiro

    2015-01-20

    Ubiquitin is known to be one of the most soluble and stably folded intracellular proteins, but it is often found in inclusion bodies associated with various diseases including neurodegenerative disorders and cancer. To gain insight into this contradictory behaviour, we have examined the physicochemical properties of ubiquitin and its polymeric chains that lead to aggregate formation. We find that the folding stability of ubiquitin chains unexpectedly decreases with increasing chain length, resulting in the formation of amyloid-like fibrils. Furthermore, when expressed in cells, polyubiquitin chains covalently linked to EGFP also form aggregates depending on chain length. Notably, these aggregates are selectively degraded by autophagy. We propose a novel model in which the physical and chemical instability of polyubiquitin chains drives the formation of fibrils, which then serve as an initiation signal for autophagy.

  8. E3 ubiquitin ligase Cullin4B mediated polyubiquitination of p53 for its degradation.

    PubMed

    Thirunavukarasou, Anand; Singh, Prachi; Govindarajalu, Gokulapriya; Bandi, Venkateshwarlu; Baluchamy, Sudhakar

    2014-05-01

    Controlled protein ubiquitination through E3 ubiquitin ligases and degradation via 26S proteasome machinery is required for orderly progression through cell cycle, chromatin remodeling, DNA repair, and development. Each cullin-dependent ubiquitin ligase (E3) complex can recruit various substrates for their degradation. Cullin 4A (CUL4A) and Cullin 4B (CUL4B) are members of cullin family proteins that mediate ubiquitin dependent proteolysis. Though, these two cul4 genes are functionally redundant, Cullin 4B is not a substitute for all the Cullin 4A functions. Published report has shown that CUL4A interacts with p53 and induces its decay. Although, CUL4A has been known to control several cellular processes, little is known about CUL4B functions. Therefore, in this study, we analyzed the role of CUL4B on p53 polyubiquitination. Our stable cell line and transient transfection studies show that CUL4B indeed interacts with p53 and induces its polyubiquitination. Importantly, both CUL4A and CUL4B overexpressing cells show almost equal levels of p53 polyubiquitination. Moreover, we observed an increased level of polyubiquitination on p53 in CUL4B overexpressing stable cell line upon treatment with siRNA specific for CUL4A indicating that CUL4B plays a vital role in p53 stability. In addition, we have observed the differential expression of CUL4B in various eukaryotic cell lines and mouse tissues suggesting the important role of CUL4B in various tissues. Together, these observations establish an important negative regulatory role of CUL4B on p53 stability.

  9. Dispersed sites of HIV Vif-dependent polyubiquitination in the DNA deaminase APOBEC3F

    PubMed Central

    Albin, John S.; Anderson, John S.; Johnson, Jeffrey R.; Harjes, Elena; Matsuo, Hiroshi; Krogan, Nevan J.; Harris, Reuben S.

    2013-01-01

    APOBEC3F and APOBEC3G are DNA cytosine deaminases that potently restrict Human Immunodeficiency Virus-type 1 replication when the virus is deprived of its accessory protein Vif. Vif counteracts these restriction factors by recruiting APOBEC3F and APOBEC3G to an E3 ubiquitin ligase complex that mediates their polyubiquitination and proteasomal degradation. While previous efforts have identified single amino acid residues in APOBEC3 proteins required for Vif recognition, less is known about the downstream ubiquitin acceptor sites that are targeted. One prior report identified a cluster of polyubiquitinated residues in APOBEC3G and proposed an antiparallel model of APOBEC3G interaction with the Vif-E3 ubiquitin ligase complex wherein Vif binding at one terminus of APOBEC3G orients the opposite terminus for polyubiquitination [Iwatani Y, et al. (2009) PNAS 106(46):19539–19544]. To test the generalizability of this model, we carried out a complete mutagenesis of the lysine residues in APOBEC3F and used a complementary, unbiased proteomic approach to identify ubiquitin acceptor sites targeted by Vif. Our data indicate that internal lysines are the dominant ubiquitin acceptor sites in both APOBEC3F and APOBEC3G. In contrast with the proposed antiparallel model, however, we find that the Vif-dependent polyubiquitination of APOBEC3F and APOBEC3G can occur at multiple acceptor sites dispersed along predicted lysine-enriched surfaces of both the N- and C-terminal deaminase domains. These data suggest an alternative model for binding of APOBEC3 proteins to the Vif-E3 ubiquitin ligase complex and diminish enthusiasm for the amenability of APOBEC3 ubiquitin acceptor sites to therapeutic intervention. PMID:23318957

  10. Inhibiting K63 Polyubiquitination Abolishes No-Go Type Stalled Translation Surveillance in Saccharomyces cerevisiae

    PubMed Central

    Saito, Kazuki; Horikawa, Wataru; Ito, Koichi

    2015-01-01

    Incidental ribosome stalling during translation elongation is an aberrant phenomenon during protein synthesis and is subjected to quality control by surveillance systems, in which mRNA and a nascent protein are rapidly degraded. Their detailed molecular mechanisms as well as responsible factors for these processes are beginning to be understood. However, the initial processes for detecting stalled translation that result in degradation remain to be determined. Among the factors identified to date, two E3 ubiquitin ligases have been reported to function in distinct manners. Because ubiquitination is one of the most versatile of cellular signals, these distinct functions of E3 ligases suggested diverse ubiquitination pathways during surveillance for stalled translation. In this study, we report experimental evidences for a unique role of non-proteasomal K63 polyubiquitination during quality control for stalled translation. Inhibiting K63 polyubiquitination by expressing a K63R ubiquitin mutation in Saccharomyces cerevisiae cells markedly abolished the quality control responses for stalled translation. More detailed analyses indicated that the effects of K63R mutants were independent of the proteasome and that K63 polyubiquitination is dependent on Hel2, one of the E3 ligases. Moreover, a K63R ubiquitin mutant barely inhibited the quality control pathway for nonstop translation, indicating distinct mechanisms for these highly related quality control pathways. Our results suggest that non-proteasomal K63 polyubiquitination is included in the initial surveillance process of stalled translation and presumably triggers protein degradation steps upon translational stall. These findings provide crucial information regarding the detailed molecular mechanisms for the initial steps involved in quality control systems and their classification. PMID:25909477

  11. Monoubiquitination of Tob/BTG family proteins competes with degradation-targeting polyubiquitination

    SciTech Connect

    Suzuki, Toru; Kim, Minsoo; Kozuka-Hata, Hiroko; Watanabe, Masato; Oyama, Masaaki; Tsumoto, Kouhei; Yamamoto, Tadashi

    2011-05-27

    Highlights: {yields} Tob/BTG family proteins are monoubiquitinated in the absence of E3s in vitro. {yields} Monoubiquitination sites of Tob are identified by mass spectrometry. {yields} The monoubiquitination event correlates with lower levels of polyubiquitination. -- Abstract: Tob belongs to the anti-proliferative Tob/BTG protein family. The expression level of Tob family proteins is strictly regulated both transcriptionally and through post-translational modification. Ubiquitin (Ub)/proteosome-dependent degradation of Tob family proteins is critical in controlling cell cycle progression and DNA damage responses. Various Ub ligases (E3s) are responsible for degradation of Tob protein. Here, we show that Tob family proteins undergo monoubiquitination even in the absence of E3s in vitro. Determination of the ubiquitination site(s) in Tob by mass spectrometric analysis revealed that two lysine residues (Lys48 and Lys63) located in Tob/BTG homology domain are ubiquitinated. A mutant Tob, in which both Lys48 and Lys63 are substituted with alanine, is more strongly polyubiquitinated than wild-type Tob in vivo. These data suggest that monoubiquitination of Tob family proteins confers resistance against polyubiquitination, which targets proteins for degradation. The strategy for regulating the stability of Tob family proteins suggests a novel role for monoubiquitination.

  12. The Proteasome Distinguishes between Heterotypic and Homotypic Lysine-11-Linked Polyubiquitin Chains.

    PubMed

    Grice, Guinevere L; Lobb, Ian T; Weekes, Michael P; Gygi, Steven P; Antrobus, Robin; Nathan, James A

    2015-07-28

    Proteasome-mediated degradation occurs with proteins principally modified with lysine-48 polyubiquitin chains. Whether the proteasome also can bind atypical ubiquitin chains, including those linked by lysine-11, has not been well established. This is critically important, as lysine-11 polyubiquitination has been implicated in both proteasome-mediated degradation and non-degradative outcomes. Here we demonstrate that pure homotypic lysine-11-linked chains do not bind strongly to the mammalian proteasome. By contrast, heterotypic polyubiquitin chains, containing lysine-11 and lysine-48 linkages, not only bind to the proteasome but also stimulate the proteasomal degradation of the cell-cycle regulator cyclin B1. Thus, while heterotypic lysine-11-linked chains facilitate proteasomal degradation, homotypic lysine-11 linkages adopt conformations that prevent association with the proteasome. Our data demonstrate the capacity of the proteasome to bind ubiquitin chains of distinct topology, with implications for the recognition and diverse biological functions of mixed ubiquitin chains. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  13. Walnut diet reduces accumulation of polyubiquitinated proteins and inflammation in the brain of aged rats.

    PubMed

    Poulose, Shibu M; Bielinski, Donna F; Shukitt-Hale, Barbara

    2013-05-01

    An increase in the aggregation of misfolded/damaged polyubiquitinated proteins has been the hallmark of many age-related neurodegenerative diseases. The accumulation of these potentially toxic proteins in brain increases with age, in part due to increased oxidative and inflammatory stresses. Walnuts, rich in omega fatty acids, have been shown to improve memory, cognition and neuronal effects related to oxidative stress (OS) and inflammation (INF) in animals and human trials. The current study found that feeding 19-month-old rats with a 6% or 9% walnut diet significantly reduced the aggregation of polyubiquitinated proteins and activated autophagy, a neuronal housekeeping function, in the striatum and hippocampus. Walnut-fed animals exhibited up-regulation of autophagy through inhibiting phosphorylation of mTOR, up-regulating ATG7 and Beclin 1, and turnover of MAP1BLC3 proteins. The clearance of polyubiquitinated protein aggregates such as p62/SQSTM1 was more profound in hippocampus, a critical region in the brain involved in memory and cognitive performance, than striatum. The clearance of ubiquitinated aggregates was in tandem with significant reductions in OS/INF, as indicated by the levels of P38-MAP kinase and phosphorylations of nuclear factor kappa B and cyclic AMP response element binding protein. The results demonstrate the effectiveness of a walnut-supplemented diet in activating the autophagy function in brain beyond its traditionally known antioxidant and anti-inflammatory benefits. Published by Elsevier Inc.

  14. VHS domains of ESCRT-0 cooperate in high-avidity binding to polyubiquitinated cargo

    SciTech Connect

    Ren, Xuefeng; Hurley, James H.

    2010-03-30

    VHS (Vps27, Hrs, and STAM) domains occur in ESCRT-0 subunits Hrs and STAM, GGA adapters, and other trafficking proteins. The structure of the STAM VHS domain-ubiquitin complex was solved at 2.6 {angstrom} resolution, revealing that determinants for ubiquitin recognition are conserved in nearly all VHS domains. VHS domains from all classes of VHS-domain containing proteins in yeast and humans, including both subunits of ESCRT-0, bound ubiquitin in vitro. ESCRTs have been implicated in the sorting of Lys63-linked polyubiquitinated cargo. Intact human ESCRT-0 binds Lys63-linked tetraubiquitin 50-fold more tightly than monoubiquitin, though only 2-fold more tightly than Lys48-linked tetraubiquitin. The gain in affinity is attributed to the cooperation of flexibly connected VHS and UIM motifs of ESCRT-0 in avid binding to the polyubiquitin chain. Mutational analysis of all the five ubiquitin-binding sites in yeast ESCRT-0 shows that cooperation between them is required for the sorting of the Lys63-linked polyubiquitinated cargo Cps1 to the vacuole.

  15. Sequential E2s drive polyubiquitin chain assembly on APC targets.

    PubMed

    Rodrigo-Brenni, Monica C; Morgan, David O

    2007-07-13

    The anaphase-promoting complex (APC), or cyclosome, is an E3 ubiquitin-protein ligase that collaborates with E2 ubiquitin-conjugating enzymes to assemble polyubiquitin chains on proteins important for cell-cycle progression. It remains unclear how the APC - or many other E3s - promotes the multiple distinct reactions necessary for chain assembly. We addressed this problem by analyzing APC interactions with different E2s. We screened all budding yeast E2s as APC coenzymes in vitro and found that two, Ubc4 and Ubc1, are the key E2 partners for the APC. These proteins display strikingly different but complementary enzymatic behaviors: Ubc4 supports the rapid monoubiquitination of multiple lysines on APC targets, while Ubc1 catalyzes K48-linked polyubiquitin chain assembly on preattached ubiquitins. Mitotic APC function is lost in yeast strains lacking both Ubc1 and Ubc4. E2-25K, a human homolog of Ubc1, also promotes APC-dependent chain extension on preattached ubiquitins. We propose that sequential E2 proteins catalyze K48-linked polyubiquitination and thus proteasomal destruction of APC targets.

  16. Polyubiquitin serves as a recognition signal, rather than a ratcheting molecule, during retrotranslocation of proteins across the endoplasmic reticulum membrane.

    PubMed

    Flierman, Dennis; Ye, Yihong; Dai, Min; Chau, Vincent; Rapoport, Tom A

    2003-09-12

    Polyubiquitination is required for retrotranslocation of proteins from the endoplasmic reticulum back into the cytosol, where they are degraded by the proteasome. We have tested whether the release of a polypeptide chain into the cytosol is caused by a ratcheting mechanism in which the attachment of polyubiquitin prevents the chain from moving back into the endoplasmic reticulum. Using a permeabilized cell system in which major histocompatibility complex class I heavy chains are retrotranslocated under the influence of the human cytomegalovirus protein US11, we demonstrate that polyubiquitination alone is insufficient to provide the driving force for retrotranslocation. Substrate release into the cytosol requires an additional ATP-dependent step. Release requires a lysine 48 linkage of ubiquitin chains. It does not occur when polyubiquitination of the substrate is carried out with glutathione S-transferase (GST)-ubiquitin, and this correlates with poly-GST-ubiquitin not being recognized by a ubiquitin-binding domain in the Ufd1-Npl4 cofactor of the ATPase p97. These data suggest that polyubiquitin does not serve as a ratcheting molecule. Rather, it may serve as a recognition signal for the p97-Ufd1-Npl4 complex, a component implicated in the movement of substrate into the cytosol.

  17. Inclusion bodies enriched for p62 and polyubiquitinated proteins in macrophages protect against atherosclerosis.

    PubMed

    Sergin, Ismail; Bhattacharya, Somashubhra; Emanuel, Roy; Esen, Emel; Stokes, Carl J; Evans, Trent D; Arif, Batool; Curci, John A; Razani, Babak

    2016-01-05

    Autophagy is a catabolic cellular mechanism that degrades dysfunctional proteins and organelles. Atherosclerotic plaque formation is enhanced in mice with macrophages deficient for the critical autophagy protein ATG5. We showed that exposure of macrophages to lipids that promote atherosclerosis increased the abundance of the autophagy chaperone p62 and that p62 colocalized with polyubiquitinated proteins in cytoplasmic inclusions, which are characterized by insoluble protein aggregates. ATG5-null macrophages developed further p62 accumulation at the sites of large cytoplasmic ubiquitin-positive inclusion bodies. Aortas from atherosclerotic mice and plaques from human endarterectomy samples showed increased abundance of p62 and polyubiquitinated proteins that colocalized with plaque macrophages, suggesting that p62-enriched protein aggregates were characteristic of atherosclerosis. The formation of the cytoplasmic inclusions depended on p62 because lipid-loaded p62-null macrophages accumulated polyubiquitinated proteins in a diffuse cytoplasmic pattern. Lipid-loaded p62-null macrophages also exhibited increased secretion of interleukin-1β (IL-1β) and had an increased tendency to undergo apoptosis, which depended on the p62 ubiquitin-binding domain and at least partly involved p62-mediated clearance of NLRP3 inflammasomes. Consistent with our in vitro observations, p62-deficient mice formed greater numbers of more complex atherosclerotic plaques, and p62 deficiency further increased atherosclerotic plaque burden in mice with a macrophage-specific ablation of ATG5. Together, these data suggested that sequestration of cytotoxic ubiquitinated proteins by p62 protects against atherogenesis, a condition in which the clearance of protein aggregates is disrupted. Copyright © 2016, American Association for the Advancement of Science.

  18. Ubiquitin expression in Neurospora crassa: cloning and sequencing of a polyubiquitin gene.

    PubMed Central

    Taccioli, G E; Grotewold, E; Aisemberg, G O; Judewicz, N D

    1989-01-01

    We have cloned and sequenced a polyubiquitin gene from Neurospora crassa that is organized in a four repeat-tandem array. The first repeat contains a small intron and the last is fused to an extra glutamine codon. In Northern blots, two RNA species of 1.3 kb and 0.7 kb hybridize to the isolated clone. The larger ubiquitin (UBI) transcript accumulates after partial inhibition of protein synthesis with cycloheximide, and the smaller one preferentially accumulates in conidia after germination. Unexpectedly, constitutive expression of UBI transcripts in exponentially grown mycelia is not altered by heat-shock or exposure to arsenite. Images PMID:2549509

  19. Automation of High-Throughput Mass Spectrometry-Based Plasma N-Glycome Analysis with Linkage-Specific Sialic Acid Esterification.

    PubMed

    Bladergroen, Marco R; Reiding, Karli R; Hipgrave Ederveen, Agnes L; Vreeker, Gerda C M; Clerc, Florent; Holst, Stephanie; Bondt, Albert; Wuhrer, Manfred; van der Burgt, Yuri E M

    2015-09-04

    Glycosylation is a post-translational modification of key importance with heterogeneous structural characteristics. Previously, we have developed a robust, high-throughput MALDI-TOF-MS method for the comprehensive profiling of human plasma N-glycans. In this approach, sialic acid residues are derivatized with linkage-specificity, namely the ethylation of α2,6-linked sialic acid residues with parallel lactone formation of α2,3-linked sialic acids. In the current study, this procedure was used as a starting point for the automation of all steps on a liquid-handling robot system. This resulted in a time-efficient and fully standardized procedure with throughput times of 2.5 h for a first set of 96 samples and approximately 1 h extra for each additional sample plate. The mass analysis of the thus-obtained glycans was highly reproducible in terms of relative quantification, with improved interday repeatability as compared to that of manual processing.

  20. Antithyroglobulin antibody

    MedlinePlus

    Thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Hypothyroidism - thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Graves disease - thyroglobulin antibody; Underactive thyroid - thyroglobulin antibody

  1. Linkage via K27 bestows ubiquitin chains with unique properties among polyubiquitins

    PubMed Central

    Castañeda, Carlos A.; Dixon, Emma K.; Walker, Olivier; Chaturvedi, Apurva; Nakasone, Mark A.; Curtis, Joseph E.; Reed, Megan R.; Krueger, Susan; Cropp, T. Ashton; Fushman, David

    2016-01-01

    Polyubiquitination, a critical protein post-translational modification, signals for a diverse set of cellular events via the different isopeptide linkages formed between C-terminus of one ubiquitin (Ub) and the ε-amine of K6, K11, K27, K29, K33, K48, or K63 of a second Ub. We assembled di-ubiquitins (Ub2) comprised of every lysine linkage and examined them biochemically and structurally. Of these, K27-Ub2 is unique as it is not cleaved by most deubiquitinases. As this remains the only structurally uncharacterized lysine-linkage, we comprehensively examined the structures and dynamics of K27-Ub2 using NMR, small-angle neutron scattering, and in silico ensemble modeling. Our structural data provide insights into functional properties of K27-Ub2, in particular, that K27-Ub2 may be specifically recognized by K48-selective receptor UBA2 domain from proteasomal shuttle protein hHR23a. Binding studies and mutagenesis confirmed this prediction, further highlighting structural/recognition versatility of polyubiquitins and the potential power of determining function from elucidation of conformational ensembles. PMID:26876099

  2. Monoubiquitination of Tob/BTG family proteins competes with degradation-targeting polyubiquitination.

    PubMed

    Suzuki, Toru; Kim, Minsoo; Kozuka-Hata, Hiroko; Watanabe, Masato; Oyama, Masaaki; Tsumoto, Kouhei; Yamamoto, Tadashi

    2011-05-27

    Tob belongs to the anti-proliferative Tob/BTG protein family. The expression level of Tob family proteins is strictly regulated both transcriptionally and through post-translational modification. Ubiquitin (Ub)/proteosome-dependent degradation of Tob family proteins is critical in controlling cell cycle progression and DNA damage responses. Various Ub ligases (E3s) are responsible for degradation of Tob protein. Here, we show that Tob family proteins undergo monoubiquitination even in the absence of E3s in vitro. Determination of the ubiquitination site(s) in Tob by mass spectrometric analysis revealed that two lysine residues (Lys48 and Lys63) located in Tob/BTG homology domain are ubiquitinated. A mutant Tob, in which both Lys48 and Lys63 are substituted with alanine, is more strongly polyubiquitinated than wild-type Tob in vivo. These data suggest that monoubiquitination of Tob family proteins confers resistance against polyubiquitination, which targets proteins for degradation. The strategy for regulating the stability of Tob family proteins suggests a novel role for monoubiquitination.

  3. Isolation of a polyubiquitin promoter and its expression in transgenic potato plants.

    PubMed Central

    Garbarino, J E; Oosumi, T; Belknap, W R

    1995-01-01

    A polyubiquitin clone (ubi7) was isolated from a potato (Solanum tuberosum) genomic library using a copy-specific probe from a stress-induced ubiquitin cDNA. The genomic clone contained a 569-bp intron immediately 5' to the initiation codon for the first ubiquitin-coding unit. Two chimeric beta-glucuronidase (GUS) fusion transgenes were introduced into potato. The first contained GUS fused to a 1156-bp promoter fragment containing only 5' flanking and 5' untranslated sequences from ubi7. The second transgene contained GUS translationally fused to the carboxy terminus of the first ubiquitin-coding unit and thus included the intron present in the 5' untranslated region of the polyubiquitin gene. Both ubi7-GUS transgenes were activated by wounding in tuber tissue and in leaves by application of exogenous methyl jasmonate. They were also expressed constitutively in the potato tuber peel (outer 1-2 mm). Both transgenes were actively expressed in mature leaves. Exceptionally high levels of expression were observed in senescent leaves. Transgenic clones containing the ubi7 intron and the first ubiquitin-coding unit showed GUS expression levels at least 10 times higher than clones containing GUS fused to the intronless promoter. PMID:8539296

  4. Isolation of a polyubiquitin promoter and its expression in transgenic potato plants.

    PubMed

    Garbarino, J E; Oosumi, T; Belknap, W R

    1995-12-01

    A polyubiquitin clone (ubi7) was isolated from a potato (Solanum tuberosum) genomic library using a copy-specific probe from a stress-induced ubiquitin cDNA. The genomic clone contained a 569-bp intron immediately 5' to the initiation codon for the first ubiquitin-coding unit. Two chimeric beta-glucuronidase (GUS) fusion transgenes were introduced into potato. The first contained GUS fused to a 1156-bp promoter fragment containing only 5' flanking and 5' untranslated sequences from ubi7. The second transgene contained GUS translationally fused to the carboxy terminus of the first ubiquitin-coding unit and thus included the intron present in the 5' untranslated region of the polyubiquitin gene. Both ubi7-GUS transgenes were activated by wounding in tuber tissue and in leaves by application of exogenous methyl jasmonate. They were also expressed constitutively in the potato tuber peel (outer 1-2 mm). Both transgenes were actively expressed in mature leaves. Exceptionally high levels of expression were observed in senescent leaves. Transgenic clones containing the ubi7 intron and the first ubiquitin-coding unit showed GUS expression levels at least 10 times higher than clones containing GUS fused to the intronless promoter.

  5. HTLV-1 Tax-induced NF{kappa}B activation is independent of Lys-63-linked-type polyubiquitination

    SciTech Connect

    Gohda, Jin; Irisawa, Masato; Tanaka, Yuetsu; Sato, Shintaro; Ohtani, Kiyoshi; Fujisawa, Jun-ichi; Inoue, Jun-ichiro . E-mail: jun-i@ims.u-tokyo.ac.jp

    2007-05-25

    Human T-cell leukemia virus type 1 (HTLV-1) Tax-induced activation of nuclear factor-{kappa}B (NF{kappa}B) is thought to play a critical role in T-cell transformation and onset of adult T-cell leukemia. However, the molecular mechanism of the Tax-induced NF{kappa}B activation remains unknown. One of the mitogen-activated protein kinase kinase kinses (MAP3Ks) members, TAK1, plays a critical role in cytokine-induced activation of NF{kappa}B, which involves lysine 63-linked (K63) polyubiquitination of NEMO, a noncatalytic subunit of the I{kappa}B kinase complex. Here we show that Tax induces K63 polyubiquitination of NEMO. However, TAK1 is dispensable for Tax-induced NF{kappa}B activation, and deubiquitination of the K63 polyubiquitin chain failed to block Tax-induced NF{kappa}B activation. In addition, silencing of other MAP3Ks, including MEKK1, MEKK3, NIK, and TPL-2, did not affect Tax-induced NF{kappa}B activation. These results strongly suggest that unlike cytokine signaling, Tax-induced NF{kappa}B activation does not involve K63 polyubiquitination-mediated MAP3K activation.

  6. Replication Fork Slowing and Reversal upon DNA Damage Require PCNA Polyubiquitination and ZRANB3 DNA Translocase Activity.

    PubMed

    Vujanovic, Marko; Krietsch, Jana; Raso, Maria Chiara; Terraneo, Nastassja; Zellweger, Ralph; Schmid, Jonas A; Taglialatela, Angelo; Huang, Jen-Wei; Holland, Cory L; Zwicky, Katharina; Herrador, Raquel; Jacobs, Heinz; Cortez, David; Ciccia, Alberto; Penengo, Lorenza; Lopes, Massimo

    2017-09-07

    DNA damage tolerance during eukaryotic replication is orchestrated by PCNA ubiquitination. While monoubiquitination activates mutagenic translesion synthesis, polyubiquitination activates an error-free pathway, elusive in mammals, enabling damage bypass by template switching. Fork reversal is driven in vitro by multiple enzymes, including the DNA translocase ZRANB3, shown to bind polyubiquitinated PCNA. However, whether this interaction promotes fork remodeling and template switching in vivo was unknown. Here we show that damage-induced fork reversal in mammalian cells requires PCNA ubiquitination, UBC13, and K63-linked polyubiquitin chains, previously involved in error-free damage tolerance. Fork reversal in vivo also requires ZRANB3 translocase activity and its interaction with polyubiquitinated PCNA, pinpointing ZRANB3 as a key effector of error-free DNA damage tolerance. Mutations affecting fork reversal also induced unrestrained fork progression and chromosomal breakage, suggesting fork remodeling as a global fork slowing and protection mechanism. Targeting these fork protection systems represents a promising strategy to potentiate cancer chemotherapy. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  7. The 5'UTR-intron of the Gladiolus polyubiquitin promoter GUBQ1 enhances translation efficiency in Gladiolus and Arabidopsis

    USDA-ARS?s Scientific Manuscript database

    A GUBQ1 polyubiquitin promoter isolated from Gladiolus was used for transformation of Gladiolus plants. The levels of GUS expression were determined by measuring specific activity of ß–glucuronidase, and it was found that GUS expression with the GUBQ1 promoter in callus, roots, and young leaves was...

  8. Polyubiquitin gene expression contributes to oxidative stress resistance in respiratory yeast (Saccharomyces cerevisiae).

    PubMed

    Cheng, L; Watt, R; Piper, P W

    1994-05-10

    UBI4, the polyubiquitin gene of Saccharomyces cerevisiae, is expressed at a low level in vegetative cells, yet induced strongly in response to starvation, cadmium, DNA-damaging agents and heat shock. UBI4 is also expressed at a higher basal level in cells growing by respiration as compared to glucose-repressed cells growing by fermentation. This higher UBI4 expression of respiratory cultures probably helps to counteract the greater oxidative stress of respiratory growth. The effects of inactivating UBI4 on high temperature viability are more marked with respiratory cultures. Also loss of UBI4 leads to a considerably increased rate of killing of respiring cells by hydrogen peroxide, whereas the same gene inactivation has relatively little effect on the peroxide sensitivity of cells in which mitochondrial functions are repressed. This is the first study to reveal that ubiquitin levels in cells can influence their ability to withstand oxidative stress.

  9. Polyubiquitin conjugation to NEMO by triparite motif protein 23 (TRIM23) is critical in antiviral defense

    PubMed Central

    Arimoto, Kei-ichiro; Funami, Kenji; Saeki, Yasushi; Tanaka, Keiji; Okawa, Katsuya; Takeuchi, Osamu; Akira, Shizuo; Murakami, Yoshiki; Shimotohno, Kunitada

    2010-01-01

    The rapid induction of type I IFN is a central event of the innate defense against viral infections and is tightly regulated by a number of cellular molecules. Viral components induce strong type I IFN responses through the activation of toll-like receptors (TLRs) and intracellular cytoplasmic receptors such as an RNA helicase RIG-I and/or MDA5. According to recent studies, the NF-κB essential modulator (NEMO, also called IKKγ) is crucial for this virus-induced antiviral response. However, the precise roles of signal activation by NEMO adaptor have not been elucidated. Here, we show that virus-induced IRF3 and NF-κB activation depends on the K(lys)-27-linked polyubiquitination to NEMO by the novel ubiquitin E3 ligase triparite motif protein 23 (TRIM23). Virus-induced IRF3 and NF-κB activation, as well as K27-linked NEMO polyubiquitination, were abrogated in TRIM23 knockdown cells, whereas TRIM23 knockdown had no effect on TNFα-mediated NF-κB activation. Furthermore, in NEMO-deficient mouse embryo fibroblast cells, IFN-stimulated response element-driven reporter activity was restored by ectopic expression of WT NEMO, as expected, but only partial recovery by NEMO K165/309/325/326/344R multipoints mutant on which TRIM23-mediated ubiquitin conjugation was substantially reduced. Thus, we conclude that TRIM23-mediated ubiquitin conjugation to NEMO is essential for TLR3- and RIG-I/MDA5-mediated antiviral innate and inflammatory responses. PMID:20724660

  10. Structural determinants of alternating (α1 → 4) and (α1 → 6) linkage specificity in reuteransucrase of Lactobacillus reuteri

    PubMed Central

    Meng, Xiangfeng; Pijning, Tjaard; Dobruchowska, Justyna M.; Yin, Huifang; Gerwig, Gerrit J.; Dijkhuizen, Lubbert

    2016-01-01

    The glucansucrase GTFA of Lactobacillus reuteri 121 produces an α-glucan (reuteran) with a large amount of alternating (α1 → 4) and (α1 → 6) linkages. The mechanism of alternating linkage formation by this reuteransucrase has remained unclear. GTFO of the probiotic bacterium Lactobacillus reuteri ATCC 55730 shows a high sequence similarity (80%) with GTFA of L. reuteri 121; it also synthesizes an α-glucan with (α1 → 4) and (α1 → 6) linkages, but with a clearly different ratio compared to GTFA. In the present study, we show that residues in loop977 (970DGKGYKGA977) and helix α4 (1083VSLKGA1088) are main determinants for the linkage specificity difference between GTFO and GTFA, and hence are important for the synthesis of alternating (α1 → 4) and (α1 → 6) linkages in GTFA. More remote acceptor substrate binding sites (i.e.+3) are also involved in the determination of alternating linkage synthesis, as shown by structural analysis of the oligosaccharides produced using panose and maltotriose as acceptor substrate. Our data show that the amino acid residues at acceptor substrate binding sites (+1, +2, +3…) together form a distinct physicochemical micro-environment that determines the alternating (α1 → 4) and (α1 → 6) linkages synthesis in GTFA. PMID:27748434

  11. SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-distributive deubiquitinating enzyme.

    PubMed

    Békés, Miklós; Rut, Wioletta; Kasperkiewicz, Paulina; Mulder, Monique P C; Ovaa, Huib; Drag, Marcin; Lima, Christopher D; Huang, Tony T

    2015-06-01

    Ubiquitin (Ub) and the Ub-like (Ubl) modifier interferon-stimulated gene 15 (ISG15) participate in the host defence of viral infections. Viruses, including the severe acute respiratory syndrome human coronavirus (SARS hCoV), have co-opted Ub-ISG15 conjugation pathways for their own advantage or have evolved effector proteins to counter pro-inflammatory properties of Ub-ISG15-conjugated host proteins. In the present study, we compare substrate specificities of the papain-like protease (PLpro) from the recently emerged Middle East respiratory syndrome (MERS) hCoV to the related protease from SARS, SARS PLpro. Through biochemical assays, we show that, similar to SARS PLpro, MERS PLpro is both a deubiquitinating (DUB) and a deISGylating enzyme. Further analysis of the intrinsic DUB activity of these viral proteases revealed unique differences between the recognition and cleavage specificities of polyUb chains. First, MERS PLpro shows broad linkage specificity for the cleavage of polyUb chains, whereas SARS PLpro prefers to cleave Lys48-linked polyUb chains. Secondly, MERS PLpro cleaves polyUb chains in a 'mono-distributive' manner (one Ub at a time) and SARS PLpro prefers to cleave Lys48-linked polyUb chains by sensing a di-Ub moiety as a minimal recognition element using a 'di-distributive' cleavage mechanism. The di-distributive cleavage mechanism for SARS PLpro appears to be uncommon among USP (Ub-specific protease)-family DUBs, as related USP family members from humans do not display such a mechanism. We propose that these intrinsic enzymatic differences between SARS and MERS PLpro will help to identify pro-inflammatory substrates of these viral DUBs and can guide in the design of therapeutics to combat infection by coronaviruses.

  12. SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-distributive deubiquitinating enzyme

    PubMed Central

    Békés, Miklós; Rut, Wioletta; Kasperkiewicz, Paulina; Mulder, Monique P. C.; Ovaa, Huib; Drag, Marcin; Lima, Christopher D.; Huang, Tony T.

    2015-01-01

    Ubiquitin (Ub) and the ubiquitin-like modifier interferon stimulated gene 15 (ISG15) participate in the host defense of viral infections. Viruses, including the Severe Acute Respiratory Syndrome human coronavirus (SARS hCoV), have co-opted Ub/ISG15-conjugation pathways for their own advantage or have evolved effector proteins to counter pro-inflammatory properties of Ub/ISG15-conjugated host proteins. Here, we compare substrate specificities of the papain-like protease (PLpro) from the recently emerged Middle Eastern Respiratory Syndrome (MERS) hCoV to the related protease from SARS, SARS PLpro. Through biochemical assays, we show that similar to SARS PLpro, MERS PLpro is both a deubiquitinating and a deISGylating enzyme. Further analysis of the intrinsic deubiquitinating enzyme (DUB) activity of these viral proteases revealed unique differences between the recognition and cleavage specificities of polyUb chains. First, MERS PLpro shows broad linkage specificity for the cleavage of polyUb chains, while SARS PLpro prefers to cleave Lys48-linked polyUb chains. Second, MERS PLpro cleaves polyUb chains in a “mono-distributive” manner (one Ub at a time), and SARS PLpro prefers to cleave K48-linked poly-Ub chains by sensing a di-Ub moiety as a minimal recognition element using a “di-distributive” cleavage mechanism. The di-distributive cleavage mechanism for SARS PLpro appears to be uncommon among USP-family DUBs, as related USP family members from humans do not display such a mechanism. We propose that these intrinsic enzymatic differences between SARS and MERS PLpro will help identify pro-inflammatory substrates of these viral DUBs and can guide in the design of therapeutics to combat infection by coronaviruses. PMID:25764917

  13. HTLV-1 tax stabilizes MCL-1 via TRAF6-dependent K63-linked polyubiquitination to promote cell survival and transformation.

    PubMed

    Choi, Young Bong; Harhaj, Edward William

    2014-10-01

    The human T-cell leukemia virus type 1 (HTLV-1) Tax protein hijacks the host ubiquitin machinery to activate IκB kinases (IKKs) and NF-κB and promote cell survival; however, the key ubiquitinated factors downstream of Tax involved in cell transformation are unknown. Using mass spectrometry, we undertook an unbiased proteome-wide quantitative survey of cellular proteins modified by ubiquitin in the presence of Tax or a Tax mutant impaired in IKK activation. Tax induced the ubiquitination of 22 cellular proteins, including the anti-apoptotic BCL-2 family member MCL-1, in an IKK-dependent manner. Tax was found to promote the nondegradative lysine 63 (K63)-linked polyubiquitination of MCL-1 that was dependent on the E3 ubiquitin ligase TRAF6 and the IKK complex. Tax interacted with and activated TRAF6, and triggered its mitochondrial localization, where it conjugated four carboxyl-terminal lysine residues of MCL-1 with K63-linked polyubiquitin chains, which stabilized and protected MCL-1 from genotoxic stress-induced degradation. TRAF6 and MCL-1 played essential roles in the survival of HTLV-1 transformed cells and the immortalization of primary T cells by HTLV-1. Therefore, K63-linked polyubiquitination represents a novel regulatory mechanism controlling MCL-1 stability that has been usurped by a viral oncogene to precipitate cell survival and transformation.

  14. Acetaminophen induces accumulation of functional rat CYP3A via polyubiquitination dysfunction.

    PubMed

    Santoh, Masataka; Sanoh, Seigo; Takagi, Masashi; Ejiri, Yoko; Kotake, Yaichiro; Ohta, Shigeru

    2016-02-22

    Acetaminophen (APAP) is extensively used as an analgesic and antipyretic drug. APAP is partly metabolized to N-acetyl-p-benzoquinone imine, a reactive metabolite, by cytochrome P450 (CYP) 1A2, 2E1 and 3A4. Some reports have indicated that CYP3A protein production and its metabolic activity are induced by APAP in rats in vivo. The CYP3A subfamily is believed to be transcriptionally regulated by chemical compounds. However, the mechanism underlying these responses is not completely understood. To clarify these mechanisms, we assessed the effects of APAP on CYP3A1/23 protein levels according to mRNA synthesis and protein degradation in rat hepatocyte spheroids, a model of liver tissue, in vivo. APAP induced CYP3A1/23 protein levels and metabolic activity. However, no change in CYP3A1/23 mRNA levels was observed. Moreover, APAP prolonged the half-life of CYP3A1/23 protein. CYP3A is known to be degraded via the ubiquitin-proteasome system. APAP significantly was found to decrease levels of polyubiquitinated CYP3A1/23 and glycoprotein 78, an E3 ligase of CYP3A1/23. These findings demonstrate that APAP induces accumulation of functional CYP3A protein via inhibition of protein degradation. Our findings may lead to the determination of novel drug-drug interactions with APAP.

  15. The proteasome controls presynaptic differentiation through modulation of an on-site pool of polyubiquitinated conjugates

    PubMed Central

    Pinto, Maria J.; Alves, Pedro L.; Martins, Luís; Pedro, Joana R.; Ryu, Hyun R.; Jeon, Noo Li; Taylor, Anne M.

    2016-01-01

    Differentiation of the presynaptic terminal is a complex and rapid event that normally occurs in spatially specific axonal regions distant from the soma; thus, it is believed to be dependent on intra-axonal mechanisms. However, the full nature of the local events governing presynaptic assembly remains unknown. Herein, we investigated the involvement of the ubiquitin–proteasome system (UPS), the major degradative pathway, in the local modulation of presynaptic differentiation. We found that proteasome inhibition has a synaptogenic effect on isolated axons. In addition, formation of a stable cluster of synaptic vesicles onto a postsynaptic partner occurs in parallel to an on-site decrease in proteasome degradation. Accumulation of ubiquitinated proteins at nascent sites is a local trigger for presynaptic clustering. Finally, proteasome-related ubiquitin chains (K11 and K48) function as signals for the assembly of presynaptic terminals. Collectively, we propose a new axon-intrinsic mechanism for presynaptic assembly through local UPS inhibition. Subsequent on-site accumulation of proteins in their polyubiquitinated state triggers formation of presynapses. PMID:27022091

  16. A single MIU motif of MINDY-1 recognizes K48-linked polyubiquitin chains.

    PubMed

    Kristariyanto, Yosua Adi; Abdul Rehman, Syed Arif; Weidlich, Simone; Knebel, Axel; Kulathu, Yogesh

    2017-03-01

    The eight different types of ubiquitin (Ub) chains that can be formed play important roles in diverse cellular processes. Linkage-selective recognition of Ub chains by Ub-binding domain (UBD)-containing proteins is central to coupling different Ub signals to specific cellular responses. The motif interacting with ubiquitin (MIU) is a small UBD that has been characterized for its binding to monoUb. The recently discovered deubiquitinase MINDY-1/FAM63A contains a tandem MIU repeat (tMIU) that is highly selective at binding to K48-linked polyUb. We here identify that this linkage-selective binding is mediated by a single MIU motif (MIU2) in MINDY-1. The crystal structure of MIU2 in complex with K48-linked polyubiquitin chains reveals that MIU2 on its own binds to all three Ub moieties in an open conformation that can only be accommodated by K48-linked triUb. The weak Ub binder MIU1 increases overall affinity of the tMIU for polyUb chains without affecting its linkage selectivity. Our analyses reveal new concepts for linkage selectivity and polyUb recognition by UBDs. © 2017 The Authors.

  17. Chemoenzymatic synthesis of bifunctional polyubiquitin substrates for monitoring ubiquitin chain remodeling.

    PubMed

    Trang, Vivian H; Rodgers, Margaret L; Boyle, Kevin J; Hoskins, Aaron A; Strieter, Eric R

    2014-07-21

    Covalent attachment of ubiquitin to target proteins is one of the most pervasive post-translational modifications in eukaryotes. Target proteins are often modified with polymeric ubiquitin chains of defined lengths and linkages that may further undergo dynamic changes in composition in response to cellular signals. Biochemical characterization of the enzymes responsible for building and destroying ubiquitin chains is often thwarted by the lack of methods for preparation of the appropriate substrates containing probes for biochemical or biophysical studies. We have discovered that a yeast ubiquitin C-terminal hydrolase (Yuh1) also catalyzes transamidation reactions that can be exploited to prepare site-specifically modified polyubiquitin chains produced by thiol-ene chemistry. We have used this chemoenzymatic approach to prepare dual-functionalized ubiquitin chains containing fluorophore and biotin modifications. These dual-functionalized ubiquitin chains enabled the first real-time assay of ubiquitin chain disassembly by a human deubiquitinase (DUB) enzyme by single molecule fluorescence microscopy. In summary, this work provides a powerful new tool for elucidating the mechanisms of DUBs and other ubiquitin processing enzymes.

  18. Keratin 8 limits TLR-triggered inflammatory responses through inhibiting TRAF6 polyubiquitination

    PubMed Central

    Dong, Xiao-Ming; Liu, En-Dong; Meng, Yun-Xiao; Liu, Chao; Bi, Ya-Lan; Wu, Huan-Wen; Jin, Yan-Chao; Yao, Jing-Hui; Tang, Liu-Jun; Wang, Jian; Li, Min; Zhang, Chao; Yu, Miao; Zhan, Yi-Qun; Chen, Hui; Ge, Chang-Hui; Yang, Xiao-Ming; Li, Chang-Yan

    2016-01-01

    Toll-like receptors (TLRs) have critical roles in innate immunity and inflammation and the detailed mechanisms by which TLR signaling is fine tuned remain unclear. Keratin 8 (CK8) belongs to the type II keratin family and is the major compontent of the intermediate filaments of simple or single-layered epithelia. Here we report that down-regulation of CK8 in mice enhanced TLR-mediated responses, rendering mice more susceptible to lipopolysaccharide (LPS)-induced endotoxin shock and Escherichia coli–caused septic peritonitis with reduced survival, elevated levels of inflammation cytokines and more severe tissue damage. We found that CK8 suppressed TLR-induced nuclear factor (NF)-κB activation and interacted with the adaptor tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) to prevent its polyubiquitination. Our findings demonstrate a novel role of CK8 in negative regulation of TLR/NF-κB signaling and highlight a previously unidentified nonclassical function for CK8 in limiting inflammatory responses. PMID:27586056

  19. Signaling, polyubiquitination, trafficking, and inclusions: sequestosome 1/p62's role in neurodegenerative disease.

    PubMed

    Wooten, Marie W; Hu, Xiao; Babu, J Ramesh; Seibenhener, M Lamar; Geetha, Thangiah; Paine, Michael G; Wooten, Michael C

    2006-01-01

    Aggregated misfolded proteins are hallmarks of most neurodegenerative diseases. In a chronic disease state, including pathologic situations of oxidative stress, these proteins are sequestered into inclusions. Accumulation of aggregated proteins can be prevented by chaperones, or by targeting their degradation to the UPS. If the accumulation of these proteins exceeds their degradation, they may impair the function of the proteasome. Alternatively, the function of the proteasome may be preserved by directing aggregated proteins to the autophagy-lysosome pathway for degradation. Sequestosome 1/p62 has recently been shown to interact with polyubiquitinated proteins through its UBA domain and may direct proteins to either the UPS or autophagosome. P62 is present in neuronal inclusions of individuals with Alzheimer's disease and other neurodegenerative diseases. Herein, we review p62's role in signaling, aggregation, and inclusion formation, and specifically as a possible contributor to Alzheimer's disease. The use of p62 as a potential target for the development of therapeutics and as a disease biomarker is also discussed.

  20. RNF20 promotes the polyubiquitination and proteasome-dependent degradation of AP-2α protein.

    PubMed

    Ren, Peng; Sheng, Zhifeng; Wang, Yijun; Yi, Xin; Zhou, Qiuzhi; Zhou, Jianlin; Xiang, Shuanglin; Hu, Xiang; Zhang, Jian

    2014-02-01

    Transcription factor activator protein 2α (AP-2α) is a negative regulator of adipogenesis by repressing the transcription of CCAAT/enhancer binding protein (C/EBPα) gene. During adipogenesis, AP-2α is degraded, leading to transcriptional up-regulation of C/EBPα. However, the mechanism for AP-2α degradation is not clear. Here, using immunoprecipitation assay and mass spectrometry, we identified ring finger protein 20 (RNF20) as an AP-2α-interacting protein in 3T3-L1 preadipocytes. RNF20 has been proved to be an E3 ubiquitin ligase for both histone H2B and tumor suppressor ErbB3-binding protein 1 (Ebp1). In this study, we demonstrated that RNF20 co-localized and interacted with AP-2α, and promoted its polyubiquitination and proteasome-dependent degradation. Over-expression of RNF20 inhibited the activity of AP-2α and rescued the C/EBPα expression which was inhibited by AP-2α. These results suggested that RNF20 may play roles in adipocyte differentiation by stimulating ubiquitin-proteasome-dependent degradation of AP-2α.

  1. A20 Ameliorates Intracerebral Hemorrhage–Induced Inflammatory Injury by Regulating TRAF6 Polyubiquitination

    PubMed Central

    Meng, Zhaoyou; Zhao, Ting; Zhou, Kai; Zhong, Qi; Wang, Yanchun; Xiong, Xiaoyi; Wang, Faxiang; Yang, Yuanrui; Zhu, Wenyao; Liu, Juan; Liao, Maofan; Wu, Lirong; Duan, Chunmei; Li, Jie; Gong, Qiuwen; Liu, Liang; Xiong, Ao; Yang, Meihua; Wang, Jian

    2017-01-01

    Reducing excessive inflammation is beneficial for the recovery from intracerebral hemorrhage (ICH). Here, the roles and mechanisms of A20 (TNFAIP3), an important endogenous anti-inflammatory factor, are examined in ICH. A20 expression in the PBMCs of ICH patients and an ICH mouse model was detected, and the correlation between A20 expression and neurologic deficits was analyzed. A20 expression was increased in PBMCs and was negatively related to the modified Rankin Scale score. A20 expression was also increased in mouse perihematomal tissues. A20−/− and A20-overexpressing mice were generated to further analyze A20 function. Compared with wild-type (WT) mice, A20−/− and A20-overexpressing mice showed significant increases and decreases, respectively, in hematoma volume, neurologic deficit score, mortality, neuronal degeneration, and proinflammatory factors. Moreover, WT-A20−/− parabiosis was established to explore the role of A20 in peripheral blood in ICH injury. ICH-induced damage, including brain edema, neurologic deficit score, proinflammatory factors, and neuronal apoptosis, was reduced in A20−/− parabionts compared with A20−/− mice. Finally, the interactions between TRAF6 and Ubc13 and UbcH5c were increased in A20−/− mice compared with WT mice; the opposite occurred in A20-overexpressing mice. Enhanced IκBα degradation and NF-κB activation were observed in A20−/− mice, but the results were reversed in A20-overexpressing mice. These results suggested that A20 is involved in regulating ICH-induced inflammatory injury in both the central and peripheral system and that A20 reduces ICH-induced inflammation by regulating TRAF6 polyubiquitination. Targeting A20 may thus be a promising therapeutic strategy for ICH. PMID:27986908

  2. The parallel reaction monitoring method contributes to a highly sensitive polyubiquitin chain quantification

    SciTech Connect

    Tsuchiya, Hikaru; Tanaka, Keiji Saeki, Yasushi

    2013-06-28

    Highlights: •The parallel reaction monitoring method was applied to ubiquitin quantification. •The ubiquitin PRM method is highly sensitive even in biological samples. •Using the method, we revealed that Ufd4 assembles the K29-linked ubiquitin chain. -- Abstract: Ubiquitylation is an essential posttranslational protein modification that is implicated in a diverse array of cellular functions. Although cells contain eight structurally distinct types of polyubiquitin chains, detailed function of several chain types including K29-linked chains has remained largely unclear. Current mass spectrometry (MS)-based quantification methods are highly inefficient for low abundant atypical chains, such as K29- and M1-linked chains, in complex mixtures that typically contain highly abundant proteins. In this study, we applied parallel reaction monitoring (PRM), a quantitative, high-resolution MS method, to quantify ubiquitin chains. The ubiquitin PRM method allows us to quantify 100 attomole amounts of all possible ubiquitin chains in cell extracts. Furthermore, we quantified ubiquitylation levels of ubiquitin-proline-β-galactosidase (Ub-P-βgal), a historically known model substrate of the ubiquitin fusion degradation (UFD) pathway. In wild-type cells, Ub-P-βgal is modified with ubiquitin chains consisting of 21% K29- and 78% K48-linked chains. In contrast, K29-linked chains are not detected in UFD4 knockout cells, suggesting that Ufd4 assembles the K29-linked ubiquitin chain(s) on Ub-P-βgal in vivo. Thus, the ubiquitin PRM is a novel, useful, quantitative method for analyzing the highly complicated ubiquitin system.

  3. F-Box Protein FBXO22 Mediates Polyubiquitination and Degradation of CD147 to Reverse Cisplatin Resistance of Tumor Cells

    PubMed Central

    Wu, Bo; Liu, Zhen-Yu; Cui, Jian; Yang, Xiang-Min; Jing, Lin; Zhou, Yang; Chen, Zhi-Nan; Jiang, Jian-Li

    2017-01-01

    Drug resistance remains a major clinical obstacle to successful treatment of cancer. As posttranslational modification is becoming widely recognized to affect the function of oncoproteins, targeting specific posttranslational protein modification provides an attractive strategy for anticancer drug development. CD147 is a transmembrane glycoprotein contributing to chemo-resistance of cancer cells in a variety of human malignancies. Ubiquitination is an important posttranslational modification mediating protein degradation. Degradation of oncoproteins, CD147 included, emerges as an attractive alternative for tumor inhibition. However, the ubiquitination of CD147 remains elusive. Here in this study, we found that deletion of the CD147 intracellular domain (CD147-ICD) prolonged the half-life of CD147 in HEK293T cells, and we identified that CD147-ICD interacts with FBXO22 using mass spectrometry and Western blot. Then, we demonstrated that FBXO22 mediates the polyubiquitination and degradation of CD147 by recognizing CD147-ICD. While knocking down of FBXO22 prolonged the half-life of CD147 in HEK293T cells, we found that FBXO22 regulates CD147 protein turnover in SMMC-7721, Huh-7 and A549 cells. Moreover, we found that the low level of FBXO22 contributes to the accumulation of CD147 and thereafter the cisplatin resistance of A549/DDP cells. To conclude, our study demonstrated that FBXO22 mediated the polyubiquitination and degradation of CD147 by interacting with CD147-ICD, and CD147 polyubiquitination by FBXO22 reversed cisplatin resistance of tumor cells. PMID:28117675

  4. HTLV-1 Tax Induces Formation of the Active Macromolecular IKK Complex by Generating Lys63- and Met1-Linked Hybrid Polyubiquitin Chains.

    PubMed

    Shibata, Yuri; Tokunaga, Fuminori; Goto, Eiji; Komatsu, Ginga; Gohda, Jin; Saeki, Yasushi; Tanaka, Keiji; Takahashi, Hirotaka; Sawasaki, Tatsuya; Inoue, Satoshi; Oshiumi, Hiroyuki; Seya, Tsukasa; Nakano, Hiroyasu; Tanaka, Yuetsu; Iwai, Kazuhiro; Inoue, Jun-Ichiro

    2017-01-01

    The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is crucial for the development of adult T-cell leukemia (ATL), a highly malignant CD4+ T cell neoplasm. Among the multiple aberrant Tax-induced effects on cellular processes, persistent activation of transcription factor NF-κB, which is activated only transiently upon physiological stimulation, is essential for leukemogenesis. We and others have shown that Tax induces activation of the IκB kinase (IKK) complex, which is a critical step in NF-κB activation, by generating Lys63-linked polyubiquitin chains. However, the molecular mechanism underlying Tax-induced IKK activation is controversial and not fully understood. Here, we demonstrate that Tax recruits linear (Met1-linked) ubiquitin chain assembly complex (LUBAC) to the IKK complex and that Tax fails to induce IKK activation in cells that lack LUBAC activity. Mass spectrometric analyses revealed that both Lys63-linked and Met1-linked polyubiquitin chains are associated with the IKK complex. Furthermore, treatment of the IKK-associated polyubiquitin chains with Met1-linked-chain-specific deubiquitinase (OTULIN) resulted in the reduction of high molecular weight polyubiquitin chains and the generation of short Lys63-linked ubiquitin chains, indicating that Tax can induce the generation of Lys63- and Met1-linked hybrid polyubiquitin chains. We also demonstrate that Tax induces formation of the active macromolecular IKK complex and that the blocking of Tax-induced polyubiquitin chain synthesis inhibited formation of the macromolecular complex. Taken together, these results lead us to propose a novel model in which the hybrid-chain-dependent oligomerization of the IKK complex triggered by Tax leads to trans-autophosphorylation-mediated IKK activation.

  5. HTLV-1 Tax Induces Formation of the Active Macromolecular IKK Complex by Generating Lys63- and Met1-Linked Hybrid Polyubiquitin Chains

    PubMed Central

    Tokunaga, Fuminori; Goto, Eiji; Komatsu, Ginga; Saeki, Yasushi; Tanaka, Keiji; Takahashi, Hirotaka; Sawasaki, Tatsuya; Inoue, Satoshi; Oshiumi, Hiroyuki; Seya, Tsukasa; Nakano, Hiroyasu; Tanaka, Yuetsu; Iwai, Kazuhiro

    2017-01-01

    The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is crucial for the development of adult T-cell leukemia (ATL), a highly malignant CD4+ T cell neoplasm. Among the multiple aberrant Tax-induced effects on cellular processes, persistent activation of transcription factor NF-κB, which is activated only transiently upon physiological stimulation, is essential for leukemogenesis. We and others have shown that Tax induces activation of the IκB kinase (IKK) complex, which is a critical step in NF-κB activation, by generating Lys63-linked polyubiquitin chains. However, the molecular mechanism underlying Tax-induced IKK activation is controversial and not fully understood. Here, we demonstrate that Tax recruits linear (Met1-linked) ubiquitin chain assembly complex (LUBAC) to the IKK complex and that Tax fails to induce IKK activation in cells that lack LUBAC activity. Mass spectrometric analyses revealed that both Lys63-linked and Met1-linked polyubiquitin chains are associated with the IKK complex. Furthermore, treatment of the IKK-associated polyubiquitin chains with Met1-linked-chain-specific deubiquitinase (OTULIN) resulted in the reduction of high molecular weight polyubiquitin chains and the generation of short Lys63-linked ubiquitin chains, indicating that Tax can induce the generation of Lys63- and Met1-linked hybrid polyubiquitin chains. We also demonstrate that Tax induces formation of the active macromolecular IKK complex and that the blocking of Tax-induced polyubiquitin chain synthesis inhibited formation of the macromolecular complex. Taken together, these results lead us to propose a novel model in which the hybrid-chain-dependent oligomerization of the IKK complex triggered by Tax leads to trans-autophosphorylation-mediated IKK activation. PMID:28103322

  6. Three Brachypodium distachyon Uev1s Promote Ubc13-Mediated Lys63-Linked Polyubiquitination and Confer Different Functions

    PubMed Central

    Guo, Huiping; Wen, Rui; Wang, Qianqian; Datla, Raju; Xiao, Wei

    2016-01-01

    In this study, we report the identification and functional characterization of three Brachypodium distachyon UEV genes. All three BdUev1s form heterodimers with BdUbc13s, which are capable of catalyzing Lys63-linked polyubiquitination in vitro. The three BdUEV1 genes are also able to functionally complement the budding yeast mms2 mutant defective in DNA-damage tolerance. BdUev1A differs from the other two BdUev1s in that it contains an 18-amino acid tail, which appears to compromise its function in yeast, as deletion of this tail restores full function. BdUev1A is excluded from the nucleus, whereas BdUev1B, BdUev1C and the C-terminally truncated BdUev1A are mainly found in the nucleus. These and the BdUEV1 gene expression analysis allow us to speculate that although all three BdUev1s function by promoting Lys63-linked polyubiquitination, BdUev1B and BdUev1C are involved in DNA-damage response and possibly other nuclear functions, while BdUev1A is required for non-nuclear function(s). PMID:27803708

  7. Lansoprazole Upregulates Polyubiquitination of the TNF Receptor-Associated Factor 6 and Facilitates Runx2-mediated Osteoblastogenesis.

    PubMed

    Mishima, Kenichi; Kitoh, Hiroshi; Ohkawara, Bisei; Okuno, Tatsuya; Ito, Mikako; Masuda, Akio; Ishiguro, Naoki; Ohno, Kinji

    2015-12-01

    The transcription factor, runt-related transcription factor 2 (Runx2), plays a pivotal role in the differentiation of the mesenchymal stem cells to the osteochondroblast lineages. We found by the drug repositioning strategy that a proton pump inhibitor, lansoprazole, enhances nuclear accumulation of Runx2 and induces osteoblastogenesis of human mesenchymal stromal cells. Systemic administration of lansoprazole to a rat femoral fracture model increased osteoblastogenesis. Dissection of signaling pathways revealed that lansoprazole activates a noncanonical bone morphogenic protein (BMP)-transforming growth factor-beta (TGF-β) activated kinase-1 (TAK1)-p38 mitogen-activated protein kinase (MAPK) pathway. We found by in cellulo ubiquitination studies that lansoprazole enhances polyubiquitination of the TNF receptor-associated factor 6 (TRAF6) and by in vitro ubiquitination studies that the enhanced polyubiquitination of TRAF6 is attributed to the blocking of a deubiquitination enzyme, cylindromatosis (CYLD). Structural modeling and site-directed mutagenesis of CYLD demonstrated that lansoprazole tightly fits in a pocket of CYLD where the C-terminal tail of ubiquitin lies. Lansoprazole is a potential therapeutic agent for enhancing osteoblastic differentiation.

  8. Natural small molecule FMHM inhibits lipopolysaccharide-induced inflammatory response by promoting TRAF6 degradation via K48-linked polyubiquitination.

    PubMed

    Zeng, Ke-Wu; Liao, Li-Xi; Lv, Hai-Ning; Song, Fang-Jiao; Yu, Qian; Dong, Xin; Li, Jun; Jiang, Yong; Tu, Peng-Fei

    2015-10-01

    TNF receptor-associated factor 6 (TRAF6) is a key hub protein involved in Toll-like receptor-dependent inflammatory signaling pathway, and it recruits additional proteins to form multiprotein complexes capable of activating downstream NF-κB inflammatory signaling pathway. Ubiquitin-proteasome system (UPS) plays a crucial role in various protein degradations, such as TRAF6, leading to inhibitory effects on inflammatory response and immunologic function. However, whether ubiquitination-dependent TRAF6 degradation can be used as a novel anti-inflammatory drug target still remains to be explored. FMHM, a bioactive natural small molecule compound extracted from Chinese herbal medicine Radix Polygalae, suppressed acute inflammatory response by targeting ubiquitin protein and inducing UPS-dependent TRAF6 degradation mechanism. It was found that FMHM targeted ubiquitin protein via Lys48 site directly induced Lys48 residue-linked polyubiquitination. This promoted Lys48 residue-linked polyubiquitin chain formation on TRAF6, resulting in increased TRAF6 degradation via UPS and inactivation of downstream NF-κB inflammatory pathway. Consequently, FMHM down-regulated inflammatory mediator levels in circulation, protected multiple organs against inflammatory injury in vivo, and prolong the survival of endotoxemia mouse models. Therefore, FMHM can serve as a novel lead compound for the development of TRAF6 scavenging agent via ubiquitination-dependent mode, which represents a promising strategy for treating inflammatory diseases.

  9. Regulation of REST levels overcomes dysregulation of neural stem cell differentiation caused by disruption of polyubiquitin gene Ubb.

    PubMed

    Kim, Minji; Ryu, Kwon-Yul

    2017-04-22

    Reduced levels of cellular ubiquitin (Ub) caused by disruption of the polyubiquitin gene Ubb lead to dysregulated differentiation of neural stem/progenitor cells (NSCs) and apoptosis in cells cultured in vitro. However, the underlying mechanisms responsible for these phenotypes in Ub-deficient cells have not been studied extensively. In the present study, we found that levels of repressor element-1 silencing transcription factor (REST) are elevated in Ubb(-/-) cells. To determine whether dysregulation of NSC differentiation is caused by the increased REST levels, we investigated the effect of reduced REST levels in Ubb(-/-) cells. Rest knockdown was found to increase the expression of the neuronal marker βIII-tubulin (TUJ1) and restore the expression pattern of the early neuronal marker α-internexin (α-INX) in Ubb(-/-) cells. Furthermore, Rest knockdown reduced Ub deficiency-induced apoptosis in cells cultured in vitro. Therefore, our study validates that cellular Ub levels are crucial for precise control of the levels of regulatory proteins such as REST during neurogenesis. We propose that regulation of Rest levels is a promising approach to overcome dysregulation of NSC differentiation caused by disruption of the polyubiquitin gene Ubb. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. TRAF1 Coordinates Polyubiquitin Signaling to Enhance Epstein-Barr Virus LMP1-Mediated Growth and Survival Pathway Activation

    PubMed Central

    Ersing, Ina; Bernhardt, Katharina; Ma, Yijie; Fu, Bishi; Ashbaugh, Camille W.; Cabo, Jackson; Mollo, Sarah B.; Zhou, Hufeng; Li, Shitao; Gewurz, Benjamin E.

    2015-01-01

    The Epstein-Barr virus (EBV) encoded oncoprotein Latent Membrane Protein 1 (LMP1) signals through two C-terminal tail domains to drive cell growth, survival and transformation. The LMP1 membrane-proximal TES1/CTAR1 domain recruits TRAFs to activate MAP kinase, non-canonical and canonical NF-kB pathways, and is critical for EBV-mediated B-cell transformation. TRAF1 is amongst the most highly TES1-induced target genes and is abundantly expressed in EBV-associated lymphoproliferative disorders. We found that TRAF1 expression enhanced LMP1 TES1 domain-mediated activation of the p38, JNK, ERK and canonical NF-kB pathways, but not non-canonical NF-kB pathway activity. To gain insights into how TRAF1 amplifies LMP1 TES1 MAP kinase and canonical NF-kB pathways, we performed proteomic analysis of TRAF1 complexes immuno-purified from cells uninduced or induced for LMP1 TES1 signaling. Unexpectedly, we found that LMP1 TES1 domain signaling induced an association between TRAF1 and the linear ubiquitin chain assembly complex (LUBAC), and stimulated linear (M1)-linked polyubiquitin chain attachment to TRAF1 complexes. LMP1 or TRAF1 complexes isolated from EBV-transformed lymphoblastoid B cell lines (LCLs) were highly modified by M1-linked polyubiqutin chains. The M1-ubiquitin binding proteins IKK-gamma/NEMO, A20 and ABIN1 each associate with TRAF1 in cells that express LMP1. TRAF2, but not the cIAP1 or cIAP2 ubiquitin ligases, plays a key role in LUBAC recruitment and M1-chain attachment to TRAF1 complexes, implicating the TRAF1:TRAF2 heterotrimer in LMP1 TES1-dependent LUBAC activation. Depletion of either TRAF1, or the LUBAC ubiquitin E3 ligase subunit HOIP, markedly impaired LCL growth. Likewise, LMP1 or TRAF1 complexes purified from LCLs were decorated by lysine 63 (K63)-linked polyubiqutin chains. LMP1 TES1 signaling induced K63-polyubiquitin chain attachment to TRAF1 complexes, and TRAF2 was identified as K63-Ub chain target. Co-localization of M1- and K63-linked

  11. Stretching of single poly-ubiquitin molecules revisited: Dynamic disorder in the non-exponential unfolding kinetics

    NASA Astrophysics Data System (ADS)

    Zheng, Yue; Bian, Yukun; Zhao, Nanrong; Hou, Zhonghuai

    2014-03-01

    A theoretical framework based on a generalized Langevin equation (GLE) with fractional Gaussian noise (fGn) and a power-law memory kernel is presented to describe the non-exponential kinetics of the unfolding of a single poly-ubiquitin molecule under a constant force [T.-L. Kuo, S. Garcia-Manyes, J. Li, I. Barel, H. Lu, B. J. Berne, M. Urbakh, J. Klafter, and J. M. Fernández, Proc. Natl. Acad. Sci. U.S.A. 107, 11336 (2010)]. Such a GLE-fGn strategy is made on the basis that the pulling coordinate variable x undergoes subdiffusion, usually resulting from conformational fluctuations, over a one-dimensional force-modified free-energy surface U(x, F). By using the Kramers' rate theory, we have obtained analytical formulae for the time-dependent rate coefficient k(t, F), the survival probability S(t, F) as well as the waiting time distribution function f(t, F) as functions of time t and force F. We find that our results can fit the experimental data of f(t, F) perfectly in the whole time range with a power-law exponent γ = 1/2, the characteristic of typical anomalous subdiffusion. In addition, the fitting of the survival probabilities for different forces facilitates us to reach rather reasonable estimations for intrinsic properties of the system, such as the free-energy barrier and the distance between the native conformation and the transition state conformation along the reaction coordinate, which are in good agreements with molecular dynamics simulations in the literatures. Although static disorder has been implicated in the original work of Kuo et al., our work suggests a sound and plausible alternative interpretation for the non-exponential kinetics in the stretching of poly-ubiquitin molecules, associated with dynamic disorder.

  12. Induction of Covalently Crosslinked p62 Oligomers with Reduced Binding to Polyubiquitinated Proteins by the Autophagy Inhibitor Verteporfin.

    PubMed

    Donohue, Elizabeth; Balgi, Aruna D; Komatsu, Masaaki; Roberge, Michel

    2014-01-01

    Autophagy is a cellular catabolic process responsible for the degradation of cytoplasmic constituents, including organelles and long-lived proteins, that helps maintain cellular homeostasis and protect against various cellular stresses. Verteporfin is a benzoporphyrin derivative used clinically in photodynamic therapy to treat macular degeneration. Verteporfin was recently found to inhibit autophagosome formation by an unknown mechanism that does not require exposure to light. We report that verteporfin directly targets and modifies p62, a scaffold and adaptor protein that binds both polyubiquitinated proteins destined for degradation and LC3 on autophagosomal membranes. Western blotting experiments revealed that exposure of cells or purified p62 to verteporfin causes the formation of covalently crosslinked p62 oligomers by a mechanism involving low-level singlet oxygen production. Rose bengal, a singlet oxygen producer structurally unrelated to verteporfin, also produced crosslinked p62 oligomers and inhibited autophagosome formation. Co-immunoprecipitation experiments demonstrated that crosslinked p62 oligomers retain their ability to bind to LC3 but show defective binding to polyubiquitinated proteins. Mutations in the p62 PB1 domain that abolish self-oligomerization also abolished crosslinked oligomer formation. Interestingly, small amounts of crosslinked p62 oligomers were detected in untreated cells, and other groups noted the accumulation of p62 forms with reduced SDS-PAGE mobility in cellular and animal models of oxidative stress and aging. These data indicate that p62 is particularly susceptible to oxidative crosslinking and lead us to propose a model whereby oxidized crosslinked p62 oligomers generated rapidly by drugs like verteporfin or over time during the aging process interfere with autophagy.

  13. A Distinct Role of Riplet-Mediated K63-Linked Polyubiquitination of the RIG-I Repressor Domain in Human Antiviral Innate Immune Responses

    PubMed Central

    Oshiumi, Hiroyuki; Miyashita, Moeko; Matsumoto, Misako; Seya, Tsukasa

    2013-01-01

    The innate immune system is essential for controlling viral infections, but several viruses have evolved strategies to escape innate immunity. RIG-I is a cytoplasmic viral RNA sensor that triggers the signal to induce type I interferon production in response to viral infection. RIG-I activation is regulated by the K63-linked polyubiquitin chain mediated by Riplet and TRIM25 ubiquitin ligases. TRIM25 is required for RIG-I oligomerization and interaction with the IPS-1 adaptor molecule. A knockout study revealed that Riplet was essential for RIG-I activation. However the molecular mechanism underlying RIG-I activation by Riplet remains unclear, and the functional differences between Riplet and TRIM25 are also unknown. A genetic study and a pull-down assay indicated that Riplet was dispensable for RIG-I RNA binding activity but required for TRIM25 to activate RIG-I. Mutational analysis demonstrated that Lys-788 within the RIG-I repressor domain was critical for Riplet-mediated K63-linked polyubiquitination and that Riplet was required for the release of RIG-I autorepression of its N-terminal CARDs, which leads to the association of RIG-I with TRIM25 ubiquitin ligase and TBK1 protein kinase. Our data indicate that Riplet is a prerequisite for TRIM25 to activate RIG-I signaling. We investigated the biological importance of this mechanism in human cells and found that hepatitis C virus (HCV) abrogated this mechanism. Interestingly, HCV NS3-4A proteases targeted the Riplet protein and abrogated endogenous RIG-I polyubiquitination and association with TRIM25 and TBK1, emphasizing the biological importance of this mechanism in human antiviral innate immunity. In conclusion, our results establish that Riplet-mediated K63-linked polyubiquitination released RIG-I RD autorepression, which allowed the access of positive factors to the RIG-I protein. PMID:23950712

  14. K63-polyubiquitinated HAUSP deubiquitinates HIF-1α and dictates H3K56 acetylation promoting hypoxia-induced tumour progression

    PubMed Central

    Wu, Han-Tsang; Kuo, Yi-Chih; Hung, Jung-Jyh; Huang, Chi-Hung; Chen, Wei-Yi; Chou, Teh-Ying; Chen, Yeh; Chen, Yi-Ju; Chen, Yu-Ju; Cheng, Wei-Chung; Teng, Shu-Chun; Wu, Kou-Juey

    2016-01-01

    Intratumoural hypoxia induces HIF-1α and promotes tumour progression, metastasis and treatment resistance. HIF-1α stability is regulated by VHL-E3 ligase-mediated ubiquitin-dependent degradation; however, the hypoxia-regulated deubiquitinase that stabilizes HIF-1α has not been identified. Here we report that HAUSP (USP7) deubiquitinase deubiquitinates HIF-1α to increase its stability, induce epithelial-mesenchymal transition and promote metastasis. Hypoxia induces K63-linked polyubiquitinated HAUSP at lysine 443 to enhance its functions. Knockdown of HAUSP decreases acetylation of histone 3 lysine 56 (H3K56Ac). K63-polyubiquitinated HAUSP interacts with a ubiquitin receptor CBP to specifically mediate H3K56 acetylation. ChIP-seq analysis of HAUSP and HIF-1α binding reveals two motifs responsive to hypoxia. HectH9 is the E3 ligase for HAUSP and a prognostic marker together with HIF-1α. This report demonstrates that hypoxia-induced K63-polyubiquitinated HAUSP deubiquitinates HIF-1α and causes CBP-mediated H3K56 acetylation on HIF-1α target gene promoters to promote EMT/metastasis, further defining HAUSP as a therapeutic target in hypoxia-induced tumour progression. PMID:27934968

  15. Ubiquitin C-terminal hydrolase-l1 activity induces polyubiquitin accumulation in podocytes and increases proteinuria in rat membranous nephropathy.

    PubMed

    Meyer-Schwesinger, Catherine; Meyer, Tobias N; Sievert, Henning; Hoxha, Elion; Sachs, Marlies; Klupp, Eva-Maria; Münster, Silvia; Balabanov, Stefan; Carrier, Lucie; Helmchen, Udo; Thaiss, Friedrich; Stahl, Rolf A K

    2011-05-01

    Ubiquitin C-terminal hydrolase L1 (UCH-L1), a key protease of the ubiquitin-proteasome system (UPS), is associated with neurodegenerative diseases and cancer. Recently, de novo expression of UCH-L1 was described in podocytes in patients with membranous nephropathy (MN), in which UCH-L1 expression correlated with increased ubiquitin content. The objective of the present study was to investigate the role of UCH-L1 in ubiquitin homeostasis and proteasomal degradation in a rat model of MN. After disease induction, UCH-L1 expression increased in podocytes and coincided with decreased glomerular monoubiquitin content. After an initial increase in proteasomal activity, the UPS was impaired. In addition to an increase of ubiquitin in podocytes, aggregates were observed 1 year after disease induction, as in MN in human beings. Inhibition of UCH-L1 hydrolase function in MN reduced UPS impairment and ameliorated proteinuria. In contrast, inhibition of proteasomal activity enhanced UPS impairment, resulting in increased proteinuria. Stable UCH-L1 overexpression in cultured podocytes resulted in accumulation of monoubiquitin and polyubiquitin proteins. In contrast, stable knock-down of UCH-L1 reduced monoubiquitin and polyubiquitin proteins and significantly increased proteasomal activity, indicating that the observed effects in rat MN also occurred in cultured podocytes. These data demonstrate that UCH-L1 activity results in polyubiquitin accumulation, proteasome inhibition, and disease aggravation in experimental models of MN.

  16. Bcl-2 Decreases the Affinity of SQSTM1/p62 to Poly-Ubiquitin Chains and Suppresses the Aggregation of Misfolded Protein in Neurodegenerative Disease.

    PubMed

    Zhou, Liang; Wang, Hongfeng; Ren, Haigang; Hu, Qingsong; Ying, Zheng; Wang, Guanghui

    2015-12-01

    Poly-ubiquitinated protein aggregate formation is the most striking hallmark of various neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, and prion disease. Mutations of many ubiquitin-associated proteins involved in the regulation of protein aggregation, such as SQSTM1/p62 (p62), parkin, and VCP, are closely linked to neurodegeneration. B-cell lymphoma 2 (Bcl-2) is a key regulator in autophagy, apoptosis, and mitochondria quality control in many cell types including neurons, and it plays important roles in the pathogenesis of neurodegenerative diseases mentioned above. Our previous work showed that Bcl-2 can directly bind to p62, and here we report that Bcl-2 directly interacts with the N-terminus of p62, but not the C-terminus (UBA domain). Interestingly and importantly, Bcl-2 affects the affinity of p62 to poly-ubiquitin chains and suppresses the aggregation of poly-ubiquitinated proteins such as mutant huntingtin associated with Huntington's disease. Our study reveals a role of Bcl-2 that involves in the regulation of misfolded proteins.

  17. Refining the minimal sequence required for ERK1/2-dependent poly-ubiquitination and proteasome-dependent turnover of BIM.

    PubMed

    Wiggins, Ceri M; Johnson, Mark; Cook, Simon J

    2010-05-01

    The pro-apoptotic protein BIM(EL) is phosphorylated by ERK1/2 and this targets the protein for poly-ubiquitination and degradation by the proteasome as a survival mechanism. To define in greater detail the sequence determinants required for BIM(EL) turnover we have compared various BIM splice variants and truncation mutants. Of the naturally occurring splice variants BIMbeta1, which lacks the C-terminal hydrophobic domain, the BH3 domain and is cytosolic, exhibited the fastest turnover rate. Indeed, neither the C-terminus, the BH3 domain nor the DLC1 binding region was required for poly-ubiquitination and turnover of BIM. However, we demonstrate that a region consisting of the ERK1/2 docking domain, ERK1/2 phosphorylation sites and either of the two potential ubiquitin-acceptor lysine residues is sufficient to allow poly-ubiquitination and turnover of BIM. In the process we demonstrate that the C-terminal hydrophobic domain, previously suggested to be important in membrane localisation, is as important as the BH3 domain for BIM to induce cell death; similarly, the pro-death BH3-domain can also confer correct mitochondrial localisation in the absence of the C-terminus. These results refine the minimal sequence for ERK1/2-driven degradation and further define the functional importance of key regions within BIM(EL), highlighting the complexity of this pro-apoptotic protein.

  18. Expression, Polyubiquitination, and Therapeutic Potential of Recombinant E6E7 from HPV16 Antigens Fused to Ubiquitin.

    PubMed

    de Oliveira, Liliane M Fernandes; Morale, Mirian G; Chaves, Agtha A M; Demasi, Marilene; Ho, Paulo L

    2017-01-01

    Ubiquitin-proteasome system plays an essential role in the immune response due to its involvement in the antigen generation and presentation to CD8(+) T cells. Hereby, ubiquitin fused to antigens has been explored as an immunotherapeutic strategy that requires the activation of cytotoxic T lymphocytes. Here we propose to apply this ubiquitin fusion approach to a recombinant vaccine against human papillomavirus 16-infected cells. E6E7 multi-epitope antigen was fused genetically at its N- or C-terminal end to ubiquitin and expressed in Escherichia coli as inclusion bodies. The antigens were solubilized using urea and purified by nickel affinity chromatography in denatured condition. Fusion of ubiquitin to E6E7 resulted in marked polyubiquitination in vitro mainly when fused to the E6E7 N-terminal. When tested in a therapeutic scenario, the fusion of ubiquitin to E6E7 reinforced the anti-tumor protection and increased the E6/E7-specific cellular immune responses. Present results encourage the investigation of the adjuvant potential of the ubiquitin fusion to recombinant vaccines requiring CD8(+) T cells.

  19. Antimitochondrial antibody

    MedlinePlus

    ... page: //medlineplus.gov/ency/article/003529.htm Antimitochondrial antibody To use the sharing features on this page, please enable JavaScript. Antimitochondrial antibodies (AMA) are substances ( antibodies ) that form against mitochondria. ...

  20. The Blue Light-Dependent Polyubiquitination and Degradation of Arabidopsis Cryptochrome2 Requires Multiple E3 Ubiquitin Ligases.

    PubMed

    Liu, Qing; Wang, Qin; Liu, Bin; Wang, Wei; Wang, Xu; Park, Joon; Yang, Zhenming; Du, Xinglin; Bian, Mingdi; Lin, Chentao

    2016-10-01

    Cryptochromes are blue light receptors regulated by light-dependent ubiquitination and degradation in both plant and animal lineages. The Arabidopsis genome encodes two cryptochromes, CRY1 and CRY2, of which CRY2 undergoes blue light-dependent ubiquitination and 26S proteasome-dependent degradation. The molecular mechanism regulating blue light-dependent proteolysis of CRY2 is still not fully understood. We found that the F-box proteins ZEITLUPE (ZTL) and Lov Kelch Protein2 (LKP2), which mediate blue light suppression of degradation of the CRY2 signaling partner CIB1, are not required for the blue light-dependent CRY2 degradation. We further showed that the previously reported function of the COP1-SPA1 protein complex in blue light-dependent CRY2 degradation is more likely to be attributable to its cullin 4 (CUL4)-based E3 ubiquitin ligase activity than its activity as the cryptochrome signaling partner. However, the blue light-dependent CRY2 degradation is only partially impaired in the cul4 mutant, the cop1-5 null mutant and the spa1234 quadruple mutant, suggesting a possible involvement of additional E3 ubiquitin ligases in the regulation of CRY2. Consistent with this hypothesis, we demonstrated that the blue light-dependent CRY2 degradation is significantly impaired in the temperature-sensitive cul1 mutant allele (axr6-3), especially under the non-permissive temperature. Based on these and other results presented, we propose that photoexcited CRY2 undergoes Lys48-linked polyubiquitination catalyzed by the CUL4- and CUL1-based E3 ubiquitin ligases.

  1. Nucleocytoplasmic Shuttling of p62/SQSTM1 and Its Role in Recruitment of Nuclear Polyubiquitinated Proteins to Promyelocytic Leukemia Bodies*

    PubMed Central

    Pankiv, Serhiy; Lamark, Trond; Bruun, Jack-Ansgar; Øvervatn, Aud; Bjørkøy, Geir; Johansen, Terje

    2010-01-01

    p62, also known as sequestosome1 (SQSTM1), A170, or ZIP, is a multifunctional protein implicated in several signal transduction pathways. p62 is induced by various forms of cellular stress, is degraded by autophagy, and acts as a cargo receptor for autophagic degradation of ubiquitinated targets. It is also suggested to shuttle ubiquitinated proteins for proteasomal degradation. p62 is commonly found in cytosolic protein inclusions in patients with protein aggregopathies, it is up-regulated in several forms of human tumors, and mutations in the gene are linked to classical adult onset Paget disease of the bone. To this end, p62 has generally been considered to be a cytosolic protein, and little attention has been paid to possible nuclear roles of this protein. Here, we present evidence that p62 shuttles continuously between nuclear and cytosolic compartments at a high rate. The protein is also found in nuclear promyelocytic leukemia bodies. We show that p62 contains two nuclear localization signals and a nuclear export signal. Our data suggest that the nucleocytoplasmic shuttling of p62 is modulated by phosphorylations at or near the most important nuclear localization signal, NLS2. The aggregation of p62 in cytosolic bodies also regulates the transport of p62 between the compartments. We found p62 to be essential for accumulation of polyubiquitinated proteins in promyelocytic leukemia bodies upon inhibition of nuclear protein export. Furthermore, p62 contributed to the assembly of proteasome-containing degradative compartments in the vicinity of nuclear aggregates containing polyglutamine-expanded Ataxin1Q84 and to the degradation of Ataxin1Q84. PMID:20018885

  2. Socs1 and Socs3 degrades Traf6 via polyubiquitination in LPS-induced acute necrotizing pancreatitis

    PubMed Central

    Zhou, X; Liu, Z; Cheng, X; Zheng, Y; Zeng, F; He, Y

    2015-01-01

    Mechanisms involved in inflammatory development during acute pancreatitis (AP) are largely vague, especially in the transformation of acute edematous pancreatitis (AEP) into acute necrotizing pancreatitis (ANP). This current study aims to investigate the functions of Traf6 in different AP models in vitro and in vivo, and to identify the possible regulatory mechanism in the progression of inflammation from mild to severe. Our data revealed that the level of Traf6 expression was significantly increased in the mild AP induced by caerulein, and the upregulation of Traf6 played a protective role in acinar cells against caerulein-induced apoptosis. In contrast, only Traf6 protein but not mRNA was downregulated in the severe ANP induced by combination treatment of caerulein and LPS. Mechanistic studies showed that LPS upregulated the levels of Socs1 and Socs3 expressions in acinar cells, Socs1 and Socs3 interacted Traf6 directly and degraded Traf6 protein via polyubiquitination, thereby counteracted the protective function of Traf6. In vivo study further showed that combination treatment of caerulein and LPS failed to induce an ANP model in the TLR4 knockout mice, and the level of Traf6 expression in the pancreatic tissues remained the same as that from the acute edematous pancreatitis (AEP) mouse. Taken together, our study reveals that Traf6 functioned as a protective factor in the progression of AP, and LPS-induced Socs1 and Socs3 exacerbate mild AP to severe AP, which provides evidence for developing a new therapeutic target to combat AP. PMID:26633718

  3. Nucleocytoplasmic shuttling of p62/SQSTM1 and its role in recruitment of nuclear polyubiquitinated proteins to promyelocytic leukemia bodies.

    PubMed

    Pankiv, Serhiy; Lamark, Trond; Bruun, Jack-Ansgar; Øvervatn, Aud; Bjørkøy, Geir; Johansen, Terje

    2010-02-19

    p62, also known as sequestosome1 (SQSTM1), A170, or ZIP, is a multifunctional protein implicated in several signal transduction pathways. p62 is induced by various forms of cellular stress, is degraded by autophagy, and acts as a cargo receptor for autophagic degradation of ubiquitinated targets. It is also suggested to shuttle ubiquitinated proteins for proteasomal degradation. p62 is commonly found in cytosolic protein inclusions in patients with protein aggregopathies, it is up-regulated in several forms of human tumors, and mutations in the gene are linked to classical adult onset Paget disease of the bone. To this end, p62 has generally been considered to be a cytosolic protein, and little attention has been paid to possible nuclear roles of this protein. Here, we present evidence that p62 shuttles continuously between nuclear and cytosolic compartments at a high rate. The protein is also found in nuclear promyelocytic leukemia bodies. We show that p62 contains two nuclear localization signals and a nuclear export signal. Our data suggest that the nucleocytoplasmic shuttling of p62 is modulated by phosphorylations at or near the most important nuclear localization signal, NLS2. The aggregation of p62 in cytosolic bodies also regulates the transport of p62 between the compartments. We found p62 to be essential for accumulation of polyubiquitinated proteins in promyelocytic leukemia bodies upon inhibition of nuclear protein export. Furthermore, p62 contributed to the assembly of proteasome-containing degradative compartments in the vicinity of nuclear aggregates containing polyglutamine-expanded Ataxin1Q84 and to the degradation of Ataxin1Q84.

  4. Disruption of polyubiquitin gene Ubc leads to defective proliferation of hepatocytes and bipotent fetal liver epithelial progenitor cells.

    PubMed

    Park, Hyejin; Yoon, Min-Sik; Ryu, Kwon-Yul

    2013-06-07

    We have previously demonstrated that disruption of polyubiquitin gene Ubc leads to mid-gestation embryonic lethality most likely due to a defect in fetal liver development, which can be partially rescued by ectopic expression of Ub. In a previous study, we assessed the cause of embryonic lethality with respect to the fetal liver hematopoietic system. We confirmed that Ubc(-/-) embryonic lethality could not be attributed to impaired function of hematopoietic stem cells, which raises the question of whether or not FLECs such as hepatocytes and bile duct cells, the most abundant cell types in the liver, are affected by disruption of Ubc and contribute to embryonic lethality. To answer this, we isolated FLCs from E13.5 embryos and cultured them in vitro. We found that proliferation capacity of Ubc(-/-) cells was significantly reduced compared to that of control cells, especially during the early culture period, however we did not observe the increased number of apoptotic cells. Furthermore, levels of Ub conjugate, but not free Ub, decreased upon disruption of Ubc expression in FLCs, and this could not be compensated for by upregulation of other poly- or mono-ubiquitin genes. Intriguingly, the highest Ubc expression levels throughout the entire culture period were observed in bipotent FLEPCs. Hepatocytes and bipotent FLEPCs were most affected by disruption of Ubc, resulting in defective proliferation as well as reduced cell numbers in vitro. These results suggest that defective proliferation of these cell types may contribute to severe reduction of fetal liver size and potentially mid-gestation lethality of Ubc(-/-) embryos. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Pellino-1 confers chemoresistance in lung cancer cells by upregulating cIAP2 through Lys63-mediated polyubiquitination

    PubMed Central

    Koh, Jaemoon; Chung, Doo Hyun

    2016-01-01

    Pellino-1 is an E3 ubiquitin ligase that mediates immune receptor signaling pathways. The role of Pellino-1 in oncogenesis of lung cancer was investigated in this study. Pellino-1 expression was increased in human lung cancer cell lines compared with non-neoplastic lung cell lines. Pellino-1 overexpression in human lung cancer cells, A549 and H1299 cells, increased the survival and colony forming ability. Pellino-1 overexpression in these cells also conferred resistance to cisplatin- or paclitaxel-induced apoptosis. In contrast, depletion of Pellino-1 decreased the survival of A549 and H1299 cells and sensitized these cells to cisplatin- and paclitaxel-induced apoptosis. Pellino-1 overexpression in A549 and H1299 cells upregulated the expression of inhibitor of apoptosis (IAP) proteins, including cIAP1 and cIAP2, while Pellino-1 depletion downregulated these molecules. Notably, Pellino-1 directly interacted with cIAP2 and stabilized cIAP2 through lysine63-mediated polyubiquitination via its E3 ligase activity. Pellino-1-mediated chemoresistance in lung cancer cells was dependent on the induction of cIAP2. Moreover, a strong positive correlation between Pellino-1 and the cIAP2 expression was observed in human lung adenocarcinoma tissues. Taken together, these results demonstrate that Pellino-1 contributes to lung oncogenesis through the overexpression of cIAP2 and promotion of cell survival and chemoresistance. Pellino-1 might be a novel oncogene and potential therapeutic target in lung cancer. PMID:27248820

  6. MID1 catalyzes the ubiquitination of protein phosphatase 2A and mutations within its Bbox1 domain disrupt polyubiquitination of alpha4 but not of PP2Ac.

    PubMed

    Du, Haijuan; Wu, Kuanlin; Didoronkute, Alma; Levy, Marcus V A; Todi, Nimish; Shchelokova, Anna; Massiah, Michael A

    2014-01-01

    MID1 is a microtubule-associated protein that belongs to the TRIM family. MID1 functions as an ubiquitin E3 ligase, and recently was shown to catalyze the polyubiquitination of, alpha4, a protein regulator of protein phosphatase 2A (PP2A). It has been hypothesized that MID1 regulates PP2A, requiring the intermediary interaction with alpha4. Here we report that MID1 catalyzes the in vitro ubiquitination of the catalytic subunit of PP2A (PP2Ac) in the absence of alpha4. In the presence of alpha4, the level of PP2Ac ubiquitination is reduced. Using the MID1 RING-Bbox1-Bbox2 (RB1B2) construct containing the E3 ligase domains, we investigate the functional effects of mutations within the Bbox domains that are identified in patients with X-linked Opitz G syndrome (XLOS). The RB1B2 proteins harboring the C142S, C145T, A130V/T mutations within the Bbox1 domain and C195F mutation within the Bbox2 domain maintain auto-polyubiquitination activity. Qualitatively, the RB1B2 proteins containing these mutations are able to catalyze the ubiquitination of PP2Ac. In contrast, the RB1B2 proteins with mutations within the Bbox1 domain are unable to catalyze the polyubiquitination of alpha4. These results suggest that unregulated alpha4 may be the direct consequence of these natural mutations in the Bbox1 domain of MID1, and hence alpha4 could play a greater role to account for the increased amount of PP2A observed in XLOS-derived fibroblasts.

  7. MID1 Catalyzes the Ubiquitination of Protein Phosphatase 2A and Mutations within Its Bbox1 Domain Disrupt Polyubiquitination of Alpha4 but Not of PP2Ac

    PubMed Central

    Du, Haijuan; Wu, Kuanlin; Didoronkute, Alma; Levy, Marcus V. A.; Todi, Nimish; Shchelokova, Anna; Massiah, Michael A.

    2014-01-01

    MID1 is a microtubule-associated protein that belongs to the TRIM family. MID1 functions as an ubiquitin E3 ligase, and recently was shown to catalyze the polyubiquitination of, alpha4, a protein regulator of protein phosphatase 2A (PP2A). It has been hypothesized that MID1 regulates PP2A, requiring the intermediary interaction with alpha4. Here we report that MID1 catalyzes the in vitro ubiquitination of the catalytic subunit of PP2A (PP2Ac) in the absence of alpha4. In the presence of alpha4, the level of PP2Ac ubiquitination is reduced. Using the MID1 RING-Bbox1-Bbox2 (RB1B2) construct containing the E3 ligase domains, we investigate the functional effects of mutations within the Bbox domains that are identified in patients with X-linked Opitz G syndrome (XLOS). The RB1B2 proteins harboring the C142S, C145T, A130V/T mutations within the Bbox1 domain and C195F mutation within the Bbox2 domain maintain auto-polyubiquitination activity. Qualitatively, the RB1B2 proteins containing these mutations are able to catalyze the ubiquitination of PP2Ac. In contrast, the RB1B2 proteins with mutations within the Bbox1 domain are unable to catalyze the polyubiquitination of alpha4. These results suggest that unregulated alpha4 may be the direct consequence of these natural mutations in the Bbox1 domain of MID1, and hence alpha4 could play a greater role to account for the increased amount of PP2A observed in XLOS-derived fibroblasts. PMID:25207814

  8. Disruption of polyubiquitin gene Ubc leads to defective proliferation of hepatocytes and bipotent fetal liver epithelial progenitor cells

    SciTech Connect

    Park, Hyejin; Yoon, Min-Sik; Ryu, Kwon-Yul

    2013-06-07

    Highlights: •Proliferation capacity of Ubc{sup −/−} FLCs was reduced during culture in vitro. •Ubc is required for proliferation of both hepatocytes and bipotent FLEPCs. •Bipotent FLEPCs exhibit highest Ubc transcription and proliferation capacity. •Cell types responsible for Ubc{sup −/−} fetal liver developmental defect were identified. -- Abstract: We have previously demonstrated that disruption of polyubiquitin gene Ubc leads to mid-gestation embryonic lethality most likely due to a defect in fetal liver development, which can be partially rescued by ectopic expression of Ub. In a previous study, we assessed the cause of embryonic lethality with respect to the fetal liver hematopoietic system. We confirmed that Ubc{sup −/−} embryonic lethality could not be attributed to impaired function of hematopoietic stem cells, which raises the question of whether or not FLECs such as hepatocytes and bile duct cells, the most abundant cell types in the liver, are affected by disruption of Ubc and contribute to embryonic lethality. To answer this, we isolated FLCs from E13.5 embryos and cultured them in vitro. We found that proliferation capacity of Ubc{sup −/−} cells was significantly reduced compared to that of control cells, especially during the early culture period, however we did not observe the increased number of apoptotic cells. Furthermore, levels of Ub conjugate, but not free Ub, decreased upon disruption of Ubc expression in FLCs, and this could not be compensated for by upregulation of other poly- or mono-ubiquitin genes. Intriguingly, the highest Ubc expression levels throughout the entire culture period were observed in bipotent FLEPCs. Hepatocytes and bipotent FLEPCs were most affected by disruption of Ubc, resulting in defective proliferation as well as reduced cell numbers in vitro. These results suggest that defective proliferation of these cell types may contribute to severe reduction of fetal liver size and potentially mid

  9. The 5′UTR-intron of the Gladiolus polyubiquitin promoter GUBQ1 enhances translation efficiency in Gladiolus and Arabidopsis

    PubMed Central

    2012-01-01

    Background There are many non-cereal monocots of agronomic, horticultural, and biofuel importance. Successful transformation of these species requires an understanding of factors controlling expression of their genes. Introns have been known to affect both the level and tissue-specific expression of genes in dicots and cereal monocots, but there have been no studies on an intron isolated from a non-cereal monocot. This study characterizes the levels of GUS expression and levels of uidA mRNA that code for β-glucuronidase (GUS) expression in leaves of Gladiolus and Arabidopsis using GUBQ1, a polyubiquitin promoter with a 1.234 kb intron, isolated from the non-cereal monocot Gladiolus, and an intronless version of this promoter. Results Gladiolus and Arabidopsis were verified by Southern hybridization to be transformed with the uidA gene that was under control of either the GUBQ1 promoter (1.9 kb), a 5′ GUBQ1 promoter missing its 1.234 kb intron (0.68 kb), or the CaMV 35 S promoter. Histochemical staining showed that GUS was expressed throughout leaves and roots of Gladiolus and Arabidopsis with the 1.9 kb GUBQ1 promoter. GUS expression was significantly decreased in Gladiolus and abolished in Arabidopsis when the 5′UTR-intron was absent. In Arabidopsis and Gladiolus, the presence of uidA mRNA was independent of the presence of the 5′UTR-intron. The 5′-UTR intron enhanced translation efficiency for both Gladiolus and Arabidopsis. Conclusions The GUBQ1 promoter directs high levels of GUS expression in young leaves of both Gladiolus and Arabidopsis. The 5′UTR-intron from GUBQ1 resulted in a similar pattern of β-glucuronidase translation efficiency for both species even though the intron resulted in different patterns of uidA mRNA accumulation for each species. PMID:22672685

  10. HTLV-1 Tax Functions as a Ubiquitin E3 Ligase for Direct IKK Activation via Synthesis of Mixed-Linkage Polyubiquitin Chains.

    PubMed

    Wang, Chong; Long, Wenying; Peng, Chao; Hu, Lin; Zhang, Qiong; Wu, Ailing; Zhang, Xiaoqing; Duan, Xiaotao; Wong, Catherine C L; Tanaka, Yuetsu; Xia, Zongping

    2016-04-01

    The HTLV-1 oncoprotein Tax plays a key role in CD4+ T cell transformation by promoting cell proliferation and survival, mainly through permanent activation of the NK-κB pathway and induction of many NF-κB target genes. Elucidating the underlying molecular mechanism is therefore critical in understanding HTLV-1-mediated transformation. Current studies have suggested multiple but controversial mechanisms regarding Tax-induced IKK activation mainly due to blending of primary Tax-induced IKK activation events and secondary IKK activation events induced by cytokines secreted by the primary Tax-induced IKK-NF-κB activation events. We reconstituted Tax-stimulated IKK activation in a cell-free system to dissect the essential cellular components for primary IKK activation by Tax and studied the underlying biochemical mechanism. We found that Tax is a putative E3 ubiquitin ligase, which, together with UbcH2, UhcH5c, or UbcH7, catalyzes the assembly of free mixed-linkage polyubiquitin chains. These free mixed-linkage polyubiquitin chains are then responsible for direct IKK activation by binding to the NEMO subunit of IKK. Our studies revealed the biochemical function of Tax in the process of IKK activation, which utilizes the minimal cellular ubiquitination components for NF-κB activation.

  11. Ubr3, a Novel Modulator of Hh Signaling Affects the Degradation of Costal-2 and Kif7 through Poly-ubiquitination

    PubMed Central

    Li, Tongchao; Giagtzoglou, Nikolaos; Lin, Guang; Jaiswal, Manish; Chen, Kuchuan; Zhang, Jie; Wei, Wei; Lewis, Michael T.; Groves, Andrew K.; Westerfield, Monte; Jia, Jianhang; Bellen, Hugo J.

    2016-01-01

    Hedgehog (Hh) signaling regulates multiple aspects of metazoan development and tissue homeostasis, and is constitutively active in numerous cancers. We identified Ubr3, an E3 ubiquitin ligase, as a novel, positive regulator of Hh signaling in Drosophila and vertebrates. Hh signaling regulates the Ubr3-mediated poly-ubiquitination and degradation of Cos2, a central component of Hh signaling. In developing Drosophila eye discs, loss of ubr3 leads to a delayed differentiation of photoreceptors and a reduction in Hh signaling. In zebrafish, loss of Ubr3 causes a decrease in Shh signaling in the developing eyes, somites, and sensory neurons. However, not all tissues that require Hh signaling are affected in zebrafish. Mouse UBR3 poly-ubiquitinates Kif7, the mammalian homologue of Cos2. Finally, loss of UBR3 up-regulates Kif7 protein levels and decreases Hh signaling in cultured cells. In summary, our work identifies Ubr3 as a novel, evolutionarily conserved modulator of Hh signaling that boosts Hh in some tissues. PMID:27195754

  12. HTLV-1 Tax Functions as a Ubiquitin E3 Ligase for Direct IKK Activation via Synthesis of Mixed-Linkage Polyubiquitin Chains

    PubMed Central

    Wang, Chong; Long, Wenying; Peng, Chao; Hu, Lin; Zhang, Qiong; Wu, Ailing; Zhang, Xiaoqing; Duan, Xiaotao; Wong, Catherine C. L.; Tanaka, Yuetsu; Xia, Zongping

    2016-01-01

    The HTLV-1 oncoprotein Tax plays a key role in CD4+ T cell transformation by promoting cell proliferation and survival, mainly through permanent activation of the NK-κB pathway and induction of many NF-κB target genes. Elucidating the underlying molecular mechanism is therefore critical in understanding HTLV-1-mediated transformation. Current studies have suggested multiple but controversial mechanisms regarding Tax-induced IKK activation mainly due to blending of primary Tax-induced IKK activation events and secondary IKK activation events induced by cytokines secreted by the primary Tax-induced IKK-NF-κB activation events. We reconstituted Tax-stimulated IKK activation in a cell-free system to dissect the essential cellular components for primary IKK activation by Tax and studied the underlying biochemical mechanism. We found that Tax is a putative E3 ubiquitin ligase, which, together with UbcH2, UhcH5c, or UbcH7, catalyzes the assembly of free mixed-linkage polyubiquitin chains. These free mixed-linkage polyubiquitin chains are then responsible for direct IKK activation by binding to the NEMO subunit of IKK. Our studies revealed the biochemical function of Tax in the process of IKK activation, which utilizes the minimal cellular ubiquitination components for NF-κB activation. PMID:27082114

  13. Ubr3, a Novel Modulator of Hh Signaling Affects the Degradation of Costal-2 and Kif7 through Poly-ubiquitination.

    PubMed

    Li, Tongchao; Fan, Junkai; Blanco-Sánchez, Bernardo; Giagtzoglou, Nikolaos; Lin, Guang; Yamamoto, Shinya; Jaiswal, Manish; Chen, Kuchuan; Zhang, Jie; Wei, Wei; Lewis, Michael T; Groves, Andrew K; Westerfield, Monte; Jia, Jianhang; Bellen, Hugo J

    2016-05-01

    Hedgehog (Hh) signaling regulates multiple aspects of metazoan development and tissue homeostasis, and is constitutively active in numerous cancers. We identified Ubr3, an E3 ubiquitin ligase, as a novel, positive regulator of Hh signaling in Drosophila and vertebrates. Hh signaling regulates the Ubr3-mediated poly-ubiquitination and degradation of Cos2, a central component of Hh signaling. In developing Drosophila eye discs, loss of ubr3 leads to a delayed differentiation of photoreceptors and a reduction in Hh signaling. In zebrafish, loss of Ubr3 causes a decrease in Shh signaling in the developing eyes, somites, and sensory neurons. However, not all tissues that require Hh signaling are affected in zebrafish. Mouse UBR3 poly-ubiquitinates Kif7, the mammalian homologue of Cos2. Finally, loss of UBR3 up-regulates Kif7 protein levels and decreases Hh signaling in cultured cells. In summary, our work identifies Ubr3 as a novel, evolutionarily conserved modulator of Hh signaling that boosts Hh in some tissues.

  14. Polyubiquitination of Transforming Growth Factor β-activated Kinase 1 (TAK1) at Lysine 562 Residue Regulates TLR4-mediated JNK and p38 MAPK Activation

    PubMed Central

    Chen, I-Ting; Hsu, Pang-Hung; Hsu, Wan-Ching; Chen, Nien-Jung; Tseng, Ping-Hui

    2015-01-01

    Toll-like receptor 4 (TLR4) plays an important role in innate immunity by eliciting inflammation. Upon receptor engagement, transforming growth factor β-activated kinase 1 (TAK1) is an essential mediator that transmits a signal from the receptor to downstream effectors, IκB kinase (IKK) and the mitogen-activated protein kinases (MAPKs), which control the production of inflammatory cytokines. However, the association between phosphorylation and ubiquitination of TAK1 is not yet clear. Here, we examined the crosstalk between phosphorylation and polyubiquitination of TAK1 and further investigated the mechanism of distinct activation of MAPKs and IKK. Inhibition of TAK1 phosphorylation enhanced Lys63-linked polyubiquitination of TAK1. Conversely, ubiquitin modification was counteracted by phospho-mimic TAK1 mutant, T(184,187)D. Moreover, using LC-MS analysis, Lys562 of TAK1 was identified as a novel Lys63-linked ubiquitination site and as the key residue in the feedback regulation. Mutation of Lys562 of TAK1 leads to a decrease in TAK1 phosphorylation and specific inhibition of the MAPK pathway, but has no effect on formation of the TAK1-containing complex. Our findings demonstrate a feedback loop for phosphorylation and ubiquitination of TAK1, indicating a dynamic regulation between TAK1 polyubiquitiantion and phosphorylated activation, and the molecular mechanism by which IKK and MAPKs are differentially activated in the TLR4 pathway. PMID:26189595

  15. En bloc transfer of polyubiquitin chains to PCNA in vitro is mediated by two different human E2–E3 pairs

    PubMed Central

    Masuda, Yuji; Suzuki, Miki; Kawai, Hidehiko; Hishiki, Asami; Hashimoto, Hiroshi; Masutani, Chikahide; Hishida, Takashi; Suzuki, Fumio; Kamiya, Kenji

    2012-01-01

    Post-replication DNA repair in eukaryotes is regulated by ubiquitination of proliferating cell nuclear antigen (PCNA). Monoubiquitination catalyzed by RAD6–RAD18 (an E2–E3 complex) stimulates translesion DNA synthesis, whereas polyubiquitination, promoted by additional factors such as MMS2–UBC13 (a UEV–E2 complex) and HLTF (an E3 ligase), leads to template switching in humans. Here, using an in vitro ubiquitination reaction system reconstituted with purified human proteins, we demonstrated that PCNA is polyubiquitinated predominantly via en bloc transfer of a pre-formed ubiquitin (Ub) chain rather than by extension of the Ub chain on monoubiquitinated PCNA. Our results support a model in which HLTF forms a thiol-linked Ub chain on UBC13 (UBC13∼Ubn) and then transfers the chain to RAD6∼Ub, forming RAD6∼Ubn+1. The resultant Ub chain is subsequently transferred to PCNA by RAD18. Thus, template switching may be promoted under certain circumstances in which both RAD18 and HLTF are coordinately recruited to sites of stalled replication. PMID:22904075

  16. Stress granule formation in Entamoeba histolytica: cross-talk between EhMLBP, EhRLE3 reverse transcriptase and polyubiquitinated proteins.

    PubMed

    Katz, Sophia; Trebicz-Geffen, Meirav; Ankri, Serge

    2014-08-01

    The Entamoeba histolytica-methylated LINE-binding protein (EhMLBP) binds to methylated repetitive DNA and is a positive regulator of a reverse transcriptase of a long interspersed nucleotide element (LINE). This protein protects trophozoites against heat shock by reducing protein aggregation. The presence of EhMLBP and polyubiquitinated proteins in heat shock-induced protein aggregates raised the question whether these proteins interact. This assumption was confirmed by co-immunoprecipitation experiments: ubiquitinated proteins were detected in the perinuclear region of non-stressed E. histolytica trophozoites, whereas ubiquitinated proteins were detected in the perinuclear region and colocalized with EhMLBP in cytoplasmic granules in heat-shocked trophozoites. We also observed that overexpression of the reverse transcriptase of EhRLE3 induced the upregulation of EhMLBP expression and the formation of these EhMLBP-containing granules. Since (i) these EhMLBP-containing granules in the cytoplasm of heat-shocked E. histolytica trophozoites also contain polyubiquitinated proteins and poly(A)(+) mRNA and (ii) their formation is promoted by sodium arsenate, puromycin, and pateamine A and is inhibited by cycloheximide, we propose that these cytoplasmic EhMLBP-containing granules are stress granules. Our data also suggest that the formation of these granules is dependent upon EhMLBP and LINE. © 2014 John Wiley & Sons Ltd.

  17. Monoclonal Antibodies.

    ERIC Educational Resources Information Center

    Killington, R. A.; Powell, K. L.

    1984-01-01

    Monoclonal antibodies have provided an exciting addition to the "armory" of the molecular biologist and immunologist. This article discusses briefly the concept of, techniques available for, production of, and possible uses of monoclonal antibodies. (Author)

  18. Monoclonal Antibodies.

    ERIC Educational Resources Information Center

    Killington, R. A.; Powell, K. L.

    1984-01-01

    Monoclonal antibodies have provided an exciting addition to the "armory" of the molecular biologist and immunologist. This article discusses briefly the concept of, techniques available for, production of, and possible uses of monoclonal antibodies. (Author)

  19. Antithyroid microsomal antibody

    MedlinePlus

    Thyroid antimicrosomal antibody; Antimicrosomal antibody; Microsomal antibody; Thyroid peroxidase antibody; TPOAb ... Granulomatous thyroiditis Hashimoto thyroiditis High levels of these antibodies have also been linked with an increased risk ...

  20. Synthetic introns help identify sequences in the 5' UTR intron of the Glycine max polyubiquitin (Gmubi) promoter that give increased promoter activity.

    PubMed

    Grant, Trudi N L; De La Torre, Carola M; Zhang, Ning; Finer, John J

    2017-04-01

    Specific sequences within the leader intron of a soybean polyubiquitin gene stimulated gene expression when placed either within a synthetic intron or upstream of a core promoter. The intron in the 5' untranslated region of the soybean polyubiquitin promoter, Gmubi, seems to contribute to the high activity of this promoter. To identify the stimulatory sequences within the intron, ten different sequential intronic sequences of 40 nt were isolated, cloned as tetrameric repeats and placed upstream of a minimal cauliflower mosaic virus 35S (35S) core promoter, which was used to control expression of the green fluorescent protein. Intron fragment tetramers were also cloned within a modified, native intron, creating a Synthetic INtron Cassette (SINC), which was then placed downstream of Gmubi and 35S core promoters. Intron fragment tetramers and SINC constructs were evaluated using transient expression in lima bean cotyledons and stable expression in soybean hairy roots. Intron fragments, used as tetramers upstream of the 35S core promoter, yielded up to 80 times higher expression than the core promoter in transient expression analyses and ten times higher expression in stably transformed hairy roots. Tetrameric intronic fragments, cloned downstream of the Gmubi and 35S core promoters and within the synthetic intron, also yielded increased transient and stable GFP expression that was up to 4 times higher than Gmubi alone and up to 40 times higher than the 35S core promoter alone. These intron fragments contain sequences that seem to act as promoter regulatory elements and may contribute to the increased expression observed with this native strong promoter. Intron regulatory elements and synthetic introns may provide additional tools for increasing transgene expression in plants.

  1. Honokiol suppresses TNF-α-induced neutrophil adhesion on cerebral endothelial cells by disrupting polyubiquitination and degradation of IκBα.

    PubMed

    Chen, Po-Jen; Wang, Yu-Ling; Kuo, Liang-Mou; Lin, Chwan-Fwu; Chen, Chun-Yu; Tsai, Yung-Fong; Shen, Jiann-Jong; Hwang, Tsong-Long

    2016-05-23

    Adhesion molecules expressed on cerebral endothelial cells (ECs) mediate leukocyte recruitment and play a significant role in cerebral inflammation. Increased levels of adhesion molecules on the EC surface induce leukocyte infiltration into inflammatory areas and are thus hallmarkers of inflammation. Honokiol, isolated from the Chinese medicinal herb Magnolia officinalis, has various pharmacological activities, including anti-inflammatory effects, yet the nature of honokiol targeting molecules remains to be revealed. Here, we investigated the inhibitory effect of honokiol on neutrophil adhesion and vascular cell adhesion molecule-1 (VCAM-1) expression, which underlie its molecular target, and mechanisms for inactivating nuclear factor κ enhancer binding protein (NF-κB) in mouse cerebral ECs. Honokiol inhibited tumour necrosis factor-α (TNF-α)-induced neutrophil adhesion and VCAM-1 gene expression in cerebral ECs. The inflammatory transcription factor NF-κB was downregulated by honokiol. Honokiol significantly blocked TNF-α-induced NF-κB p65 nuclear translocation and degradation of the proteasome-dependent inhibitor of NF-κB α (IκBα). From docking model prediction, honokiol directly targeted the ubiquitin-ubiquitin interface of Lys48-linked polychains. Moreover, honokiol prevented the TNF-α-induced Lys48-linked polyubiquitination, including IκBα-polyubiquitin interaction. Honokiol has protective anti-inflammatory effects on TNF-α-induced neutrophil adhesion and VCAM-1 gene expression in cerebral ECs, at least in part by directly inhibiting ubiquitination-mediated IκBα degradation and then preventing NF-κB nuclear translocation.

  2. Monoclonal Antibodies.

    PubMed

    Geskin, Larisa J

    2015-10-01

    Use of monoclonal antibodies (mAbs) has revolutionized cancer therapy. Approaches targeting specific cellular targets on the malignant cells and in tumor microenvironment have been proved to be successful in hematologic malignancies, including cutaneous lymphomas. mAb-based therapy for cutaneous T-cell lymphoma has demonstrated high response rates and a favorable toxicity profile in clinical trials. Several antibodies and antibody-based conjugates are approved for use in clinical practice, and many more are in ongoing and planned clinical trials. In addition, these safe and effective drugs can be used as pillars for sequential therapies in a rational stepwise manner.

  3. Complex of Fas-associated factor 1 (FAF1) with valosin-containing protein (VCP)-Npl4-Ufd1 and polyubiquitinated proteins promotes endoplasmic reticulum-associated degradation (ERAD).

    PubMed

    Lee, Jae-Jin; Park, Joon Kyu; Jeong, Jaeho; Jeon, Hyesung; Yoon, Jong-Bok; Kim, Eunice EunKyeong; Lee, Kong-Joo

    2013-03-08

    Fas-associated factor 1 (FAF1) is a ubiquitin receptor containing multiple ubiquitin-related domains including ubiquitin-associated (UBA), ubiquitin-like (UBL) 1, UBL2, and ubiquitin regulatory X (UBX). We previously showed that N-terminal UBA domain recognizes Lys(48)-ubiquitin linkage to recruit polyubiquitinated proteins and that a C-terminal UBX domain interacts with valosin-containing protein (VCP). This study shows that FAF1 interacts only with VCP complexed with Npl4-Ufd1 heterodimer, a requirement for the recruitment of polyubiquitinated proteins to UBA domain. Intriguingly, VCP association to C-terminal UBX domain regulates ubiquitin binding to N-terminal UBA domain without direct interaction between UBA and UBX domains. These interactions are well characterized by structural and biochemical analysis. VCP-Npl4-Ufd1 complex is known as the machinery required for endoplasmic reticulum-associated degradation. We demonstrate here that FAF1 binds to VCP-Npl4-Ufd1 complex via UBX domain and polyubiquitinated proteins via UBA domain to promote endoplasmic reticulum-associated degradation.

  4. Disassembly of Lys11 and Mixed Linkage Polyubiquitin Conjugates Provides Insights into Function of Proteasomal Deubiquitinases Rpn11 and Ubp6*

    PubMed Central

    Mansour, Wissam; Nakasone, Mark A.; von Delbrück, Maximilian; Yu, Zanlin; Krutauz, Daria; Reis, Noa; Kleifeld, Oded; Sommer, Thomas; Fushman, David; Glickman, Michael H.

    2015-01-01

    Protein homeostasis is largely dependent on proteolysis by the ubiquitin-proteasome system. Diverse polyubiquitin modifications are reported to target cellular proteins to the proteasome. At the proteasome, deubiquitination is an essential preprocessing event that contributes to degradation efficiency. We characterized the specificities of two proteasome-associated deubiquitinases (DUBs), Rpn11 and Ubp6, and explored their impact on overall proteasome DUB activity. This was accomplished by constructing a panel of well defined ubiquitin (Ub) conjugates, including homogeneous linkages of varying lengths as well as a heterogeneously modified target. Rpn11 and Ubp6 processed Lys11 and Lys63 linkages with comparable efficiencies that increased with chain length. In contrast, processing of Lys48 linkages by proteasome was inversely correlated to chain length. Fluorescently labeled tetra-Ub chains revealed endo-chain preference for Ubp6 acting on Lys48 and random action for Rpn11. Proteasomes were more efficient at deconjugating identical substrates than their constituent DUBs by roughly 2 orders of magnitude. Incorporation into proteasomes significantly enhanced enzymatic efficiency of Rpn11, due in part to alleviation of the autoinhibitory role of its C terminus. The broad specificity of Rpn11 could explain how proteasomes were more effective at disassembling a heterogeneously modified conjugate compared with homogeneous Lys48-linked chains. The reduced ability to disassemble homogeneous Lys48-linked chains longer than 4 Ub units may prolong residency time on the proteasome. PMID:25389291

  5. The human homologue of the yeast polyubiquitination factor Ufd2p is cleaved by caspase 6 and granzyme B during apoptosis.

    PubMed Central

    Mahoney, James A; Odin, Joseph A; White, Sarah M; Shaffer, David; Koff, Andrew; Casciola-Rosen, Livia; Rosen, Antony

    2002-01-01

    In the present study, we demonstrate that a human homologue of Ufd2p (a yeast protein that catalyses the formation of long polyubiquitin chains, and is implicated in responses to environmental stress), UFD2 (ubiquitin fusion degradation protein-2), is cleaved during apoptosis induced by multiple stimuli, including UVB irradiation, Fas ligation, staurosporine treatment and cytotoxic lymphocyte granule-induced death. Caspase 6 and granzyme B efficiently cleave UFD2 [k(cat)/K(m)=(4-5) x 10(4) M(-1) x s(-1)] at Asp(123), whereas caspases 3 and 7 cleave UFD2 approx. 10-fold less efficiently immediately upstream at Asp(109). Thus UFD2 is added to the growing list of proteins with closely spaced caspase and granzyme B cleavage sites, suggesting the presence of a previously unrecognized, conserved motif. Both cleavage sites are contained and conserved within a novel 300-amino-acid N-terminal domain present in apparent UFD2 orthologues in mice and zebrafish, but absent in all UFD2 family members in lower eukaryotes. Full-length recombinant UFD2 exhibited ubiquitin-protein ligase ('E3')-like ubiquitination activity in vitro, but this activity was abolished in recombinant UFD2 truncated at the granzyme B/caspase 6 cleavage site. Cleavage of UFD2 by caspases or granzyme B within this putative regulatory N-terminal domain might have important functional consequences within the apoptotic cascade. PMID:11802788

  6. Parkin-mediated K63-polyubiquitination targets ubiquitin C-terminal hydrolase L1 for degradation by the autophagy-lysosome system.

    PubMed

    McKeon, Jeanne E; Sha, Di; Li, Lian; Chin, Lih-Shen

    2015-05-01

    Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a key neuronal deubiquitinating enzyme which is mutated in Parkinson disease (PD) and in childhood-onset neurodegenerative disorder with optic atrophy. Furthermore, reduced UCH-L1 protein levels are associated with a number of neurodegenerative diseases, whereas up-regulation of UCH-L1 protein expression is found in multiple types of cancer. However, very little is known about how UCH-L1 protein level is regulated in cells. Here, we report that UCH-L1 is a novel interactor and substrate of PD-linked E3 ubiquitin-protein ligase parkin. We find that parkin mediates K63-linked polyubiquitination of UCH-L1 in cooperation with the Ubc13/Uev1a E2 ubiquitin-conjugating enzyme complex and promotes UCH-L1 degradation by the autophagy-lysosome pathway. Targeted disruption of parkin gene expression in mice causes a significant decrease in UCH-L1 ubiquitination with a concomitant increase in UCH-L1 protein level in brain, supporting an in vivo role of parkin in regulating UCH-L1 ubiquitination and degradation. Our findings reveal a direct link between parkin-mediated ubiquitin signaling and UCH-L1 regulation, and they have important implications for understanding the roles of these two proteins in health and disease.

  7. Antiparietal cell antibody test

    MedlinePlus

    APCA; Anti-gastric parietal cell antibody; Atrophic gastritis - anti-gastric parietal cell antibody; Gastric ulcer - anti-gastric parietal cell antibody; Pernicious anemia - anti-gastric parietal cell antibody; Vitamin B12 - anti-gastric ...

  8. Role of Nedd4-2 and polyubiquitination in epithelial sodium channel degradation in untransfected renal A6 cells expressing endogenous ENaC subunits.

    PubMed

    Malik, B; Yue, Q; Yue, G; Chen, X J; Price, S R; Mitch, W E; Eaton, D C

    2005-07-01

    Amiloride-sensitive epithelial sodium channels (ENaC) are responsible for transepithelial Na(+) transport in the kidney, lung, and colon. The channel consists of three subunits (alpha, beta, and gamma). In Madin-Darby canine kidney (MDCK) cells and Xenopus laevis oocytes transfected with all three ENaC subunits, neural precursor cell-expressed developmentally downregulated protein (Nedd4-2) promotes ubiquitin conjugation of ENaC. For native proteins in some cells, ubiquitin conjugation is a signal for their degradation by the ubiquitin-proteasome pathway, whereas in other cell types ubiquitin conjugation is a signal for endocytosis and lysosomal protein degradation. When ENaC are transfected into MDCK cells, ubiquitin conjugation leads to lysosomal degradation. In this paper, we characterize the involvement of the ubiquitin-proteasome proteolytic pathway in the regulation of functional ENaC in untransfected renal A6 cells expressing native ENaC subunits. In contrast to transfected cells, we show that total cellular alpha-, beta-, and gamma-ENaC subunits are polyubiquitinated and that ubiquitin conjugation of subunits increases when the cells are treated with a proteasome inhibitor. We show that Nedd4-2 is associated with alpha- and beta-subunits and is associated with the apical membrane. We also show the Nedd4-2 can regulate the number of functional ENaC subunits in the apical membrane. The results reported here suggest that the ubiquitin-proteasome proteolytic pathway is an important determinant of ENaC function in untransfected renal cells expressing endogenous ENaC.

  9. Phosphomimetic mutation of the N-terminal lid of MDM2 enhances the polyubiquitination of p53 through stimulation of E2-ubiquitin thioester hydrolysis.

    PubMed

    Fraser, Jennifer A; Worrall, Erin G; Lin, Yao; Landre, Vivien; Pettersson, Susanne; Blackburn, Elizabeth; Walkinshaw, Malcolm; Muller, Petr; Vojtesek, Borek; Ball, Kathryn; Hupp, Ted R

    2015-04-24

    Mouse double minute 2 (MDM2) has a phosphorylation site within a lid motif at Ser17 whose phosphomimetic mutation to Asp17 stimulates MDM2-mediated polyubiquitination of p53. MDM2 lid deletion, but not Asp17 mutation, induced a blue shift in the λ(max) of intrinsic fluorescence derived from residues in the central domain including Trp235, Trp303, Trp323, and Trp329. This indicates that the Asp17 mutation does not alter the conformation of MDM2 surrounding the tryptophan residues. In addition, Phe235 mutation enhanced MDM2 binding to p53 but did not stimulate its ubiquitination function, thus uncoupling increases in p53 binding from its E3 ubiquitin ligase function. However, the Asp17 mutation in MDM2 stimulated its discharge of the UBCH5a-ubiquitin thioester adduct (UBCH5a is a ubiquitin-conjugating enzyme E2D 1 UBC4/5 homolog yeast). This stimulation of ubiquitin discharge from E2 was independent of the p53 substrate. There are now four known effects of the Asp17 mutation on MDM2: (i) it alters the conformation of the isolated N-terminus as defined by NMR; (ii) it induces increased thermostability of the isolated N-terminal domain; (iii) it stimulates the allosteric interaction of MDM2 with the DNA-binding domain of p53; and (iv) it stimulates a novel protein-protein interaction with the E2-ubiquitin complex in the absence of substrate p53 that, in turn, increases hydrolysis of the E2-ubiquitin thioester bond. These data also suggest a new strategy to disrupt MDM2 function by targeting the E2-ubiquitin discharge reaction.

  10. The MID1 E3 ligase catalyzes the polyubiquitination of Alpha4 (α4), a regulatory subunit of protein phosphatase 2A (PP2A): novel insights into MID1-mediated regulation of PP2A.

    PubMed

    Du, Haijuan; Huang, Yongzhao; Zaghlula, Manar; Walters, Erica; Cox, Timothy C; Massiah, Michael A

    2013-07-19

    Alpha4 (α4) is a key regulator of protein phosphatase 2A (PP2A) and mTOR in steps essential for cell-cycle progression. α4 forms a complex with PP2A and MID1, a microtubule-associated ubiquitin E3 ligase that facilitates MID1-dependent regulation of PP2A and the dephosphorylation of MID1 by PP2A. Ectopic overexpression of α4 is associated with hepatocellular carcinomas, breast cancer, and invasive adenocarcinomas. Here, we provide data suggesting that α4 is regulated by ubiquitin-dependent degradation mediated by MID1. In cells stably expressing a dominant-negative form of MID1, significantly elevated levels of α4 were observed. Treatment of cells with the specific proteasome inhibitor, lactacystin, resulted in a 3-fold increase in α4 in control cells and a similar level in mutant cells. Using in vitro assays, individual MID1 E3 domains facilitated monoubiquitination of α4, whereas full-length MID1 as well as RING-Bbox1 and RING-Bbox1-Bbox2 constructs catalyzed its polyubiquitination. In a novel non-biased functional screen, we identified a leucine to glutamine substitution at position 146 within Bbox1 that abolished MID1-α4 interaction and the subsequent polyubiquitination of α4, indicating that direct binding to Bbox1 was necessary for the polyubiquitination of α4. The mutant had little impact on the RING E3 ligase functionality of MID1. Mass spectrometry data confirmed Western blot analysis that ubiquitination of α4 occurs only within the last 105 amino acids. These novel findings identify a new role for MID1 and a mechanism of regulation of α4 that is likely to impact the stability and activity level of PP2Ac.

  11. Antibody Engineering and Therapeutics

    PubMed Central

    Almagro, Juan Carlos; Gilliland, Gary L; Breden, Felix; Scott, Jamie K; Sok, Devin; Pauthner, Matthias; Reichert, Janice M; Helguera, Gustavo; Andrabi, Raiees; Mabry, Robert; Bléry, Mathieu; Voss, James E; Laurén, Juha; Abuqayyas, Lubna; Barghorn, Stefan; Ben-Jacob, Eshel; Crowe, James E; Huston, James S; Johnston, Stephen Albert; Krauland, Eric; Lund-Johansen, Fridtjof; Marasco, Wayne A; Parren, Paul WHI; Xu, Kai Y

    2014-01-01

    The 24th Antibody Engineering & Therapeutics meeting brought together a broad range of participants who were updated on the latest advances in antibody research and development. Organized by IBC Life Sciences, the gathering is the annual meeting of The Antibody Society, which serves as the scientific sponsor. Preconference workshops on 3D modeling and delineation of clonal lineages were featured, and the conference included sessions on a wide variety of topics relevant to researchers, including systems biology; antibody deep sequencing and repertoires; the effects of antibody gene variation and usage on antibody response; directed evolution; knowledge-based design; antibodies in a complex environment; polyreactive antibodies and polyspecificity; the interface between antibody therapy and cellular immunity in cancer; antibodies in cardiometabolic medicine; antibody pharmacokinetics, distribution and off-target toxicity; optimizing antibody formats for immunotherapy; polyclonals, oligoclonals and bispecifics; antibody discovery platforms; and antibody-drug conjugates. PMID:24589717

  12. Rapid Activation of Transforming Growth Factor β–Activated Kinase 1 in Chondrocytes by Phosphorylation and K63‐Linked Polyubiquitination Upon Injury to Animal Articular Cartilage

    PubMed Central

    Ismail, Heba M.; Didangelos, Athanasios; Vincent, Tonia L.

    2017-01-01

    Objective Mechanical injury to cartilage predisposes to osteoarthritis (OA). Wounding of the articular cartilage surface causes rapid activation of MAP kinases and NF‐κB, mimicking the response to inflammatory cytokines. This study was undertaken to identify the upstream signaling mechanisms involved. Methods Cartilage was injured by dissecting it from the articular surface of porcine metacarpophalangeal (MCP) joints or by avulsing murine proximal femoral epiphyses. Protein phosphorylation was assayed by Western blotting of cartilage lysates. Immunolocalization of phosphorylated activating transcription factor 2 (ATF‐2) and NF‐κB/p65 was detected by confocal microscopy. Messenger RNA (mRNA) was measured by quantitative reverse transcriptase–polymerase chain reaction (qRT‐PCR). Receptor associated protein 80 (RAP‐80) ubiquitin interacting motif agarose was used in a pull‐down assay to obtain K63‐polyubiquitinated proteins. Ubiquitin linkages on immunoprecipitated transforming growth factor β–activated kinase 1 (TAK‐1) were analyzed with deubiquitinases. Results Sharp injury to porcine cartilage caused rapid activation of JNK and NF‐κB pathways and the upstream kinases MKK‐4, IKK, and TAK‐1. Pharmacologic inhibition of TAK‐1 in porcine cartilage abolished JNK and NF‐κB activation and reduced the injury‐dependent inflammatory gene response. High molecular weight species of phosphorylated TAK‐1 were induced by injury, indicating its ubiquitination. An overall increase in K63‐linked polyubiquitination was detected upon injury, and TAK‐1 was specifically linked to K63‐ but not K48‐polyubiquitin chains. In mice, avulsion of wild‐type femoral epiphyses caused similar intracellular signaling that was reduced in cartilage‐specific TAK‐1–null mice. Epiphyseal cartilage of MyD88‐null and TRAF‐6–null mice responded to injury, suggesting the involvement of a ubiquitin E3 ligase other than TRAF‐6. Conclusion

  13. HTLV-1 Tax Stimulates Ubiquitin E3 Ligase, Ring Finger Protein 8, to Assemble Lysine 63-Linked Polyubiquitin Chains for TAK1 and IKK Activation.

    PubMed

    Ho, Yik-Khuan; Zhi, Huijun; Bowlin, Tara; Dorjbal, Batsukh; Philip, Subha; Zahoor, Muhammad Atif; Shih, Hsiu-Ming; Semmes, Oliver John; Schaefer, Brian; Glover, J N Mark; Giam, Chou-Zen

    2015-08-01

    Human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, impacts a multitude of cellular processes, including I-κB kinase (IKK)/NF-κB signaling, DNA damage repair, and mitosis. These activities of Tax have been implicated in the development of adult T-cell leukemia (ATL) in HTLV-1-infected individuals, but the underlying mechanisms remain obscure. IKK and its upstream kinase, TGFβ-activated kinase 1 (TAK1), contain ubiquitin-binding subunits, NEMO and TAB2/3 respectively, which interact with K63-linked polyubiquitin (K63-pUb) chains. Recruitment to K63-pUb allows cross auto-phosphorylation and activation of TAK1 to occur, followed by TAK1-catalyzed IKK phosphorylation and activation. Using cytosolic extracts of HeLa and Jurkat T cells supplemented with purified proteins we have identified ubiquitin E3 ligase, ring finger protein 8 (RNF8), and E2 conjugating enzymes, Ubc13:Uev1A and Ubc13:Uev2, to be the cellular factors utilized by Tax for TAK1 and IKK activation. In vitro, the combination of Tax and RNF8 greatly stimulated TAK1, IKK, IκBα and JNK phosphorylation. In vivo, RNF8 over-expression augmented while RNF8 ablation drastically reduced canonical NF-κB activation by Tax. Activation of the non-canonical NF-κB pathway by Tax, however, is unaffected by the loss of RNF8. Using purified components, we further demonstrated biochemically that Tax greatly stimulated RNF8 and Ubc13:Uev1A/Uev2 to assemble long K63-pUb chains. Finally, co-transfection of Tax with increasing amounts of RNF8 greatly induced K63-pUb assembly in a dose-dependent manner. Thus, Tax targets RNF8 and Ubc13:Uev1A/Uev2 to promote the assembly of K63-pUb chains, which signal the activation of TAK1 and multiple downstream kinases including IKK and JNK. Because of the roles RNF8 and K63-pUb chains play in DNA damage repair and cytokinesis, this mechanism may also explain the genomic instability of HTLV-1-transformed T cells and ATL cells.

  14. HTLV-1 Tax Stimulates Ubiquitin E3 Ligase, Ring Finger Protein 8, to Assemble Lysine 63-Linked Polyubiquitin Chains for TAK1 and IKK Activation

    PubMed Central

    Ho, Yik-Khuan; Zhi, Huijun; Bowlin, Tara; Dorjbal, Batsukh; Philip, Subha; Zahoor, Muhammad Atif; Shih, Hsiu-Ming; Semmes, Oliver John; Schaefer, Brian; Glover, J. N. Mark; Giam, Chou-Zen

    2015-01-01

    Human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, impacts a multitude of cellular processes, including I-κB kinase (IKK)/NF-κB signaling, DNA damage repair, and mitosis. These activities of Tax have been implicated in the development of adult T-cell leukemia (ATL) in HTLV-1-infected individuals, but the underlying mechanisms remain obscure. IKK and its upstream kinase, TGFβ-activated kinase 1 (TAK1), contain ubiquitin-binding subunits, NEMO and TAB2/3 respectively, which interact with K63-linked polyubiquitin (K63-pUb) chains. Recruitment to K63-pUb allows cross auto-phosphorylation and activation of TAK1 to occur, followed by TAK1-catalyzed IKK phosphorylation and activation. Using cytosolic extracts of HeLa and Jurkat T cells supplemented with purified proteins we have identified ubiquitin E3 ligase, ring finger protein 8 (RNF8), and E2 conjugating enzymes, Ubc13:Uev1A and Ubc13:Uev2, to be the cellular factors utilized by Tax for TAK1 and IKK activation. In vitro, the combination of Tax and RNF8 greatly stimulated TAK1, IKK, IκBα and JNK phosphorylation. In vivo, RNF8 over-expression augmented while RNF8 ablation drastically reduced canonical NF-κB activation by Tax. Activation of the non-canonical NF-κB pathway by Tax, however, is unaffected by the loss of RNF8. Using purified components, we further demonstrated biochemically that Tax greatly stimulated RNF8 and Ubc13:Uev1A/Uev2 to assemble long K63-pUb chains. Finally, co-transfection of Tax with increasing amounts of RNF8 greatly induced K63-pUb assembly in a dose-dependent manner. Thus, Tax targets RNF8 and Ubc13:Uev1A/Uev2 to promote the assembly of K63-pUb chains, which signal the activation of TAK1 and multiple downstream kinases including IKK and JNK. Because of the roles RNF8 and K63-pUb chains play in DNA damage repair and cytokinesis, this mechanism may also explain the genomic instability of HTLV-1-transformed T cells and ATL cells. PMID:26285145

  15. Oligomerization of deoxynucleoside-biphosphate dimers - Template and linkage specificity

    NASA Technical Reports Server (NTRS)

    Visscher, J.; Van Der Woerd, R.; Bakker, C. G.; Schwartz, Alan W.

    1989-01-01

    The oligomerization of the activated 3-prime-5-prime pyrophosphate-linked dimer, pdAppdAp, is presently noted to be selectively favored by a poly(U) template over the 3-prime-3-prime and 5-prime-5-prime linked dimers. Both overall yields and the production of the longest oligomers were markedly stimulated by poly(U)'s presence; in its absence, the 5-prime-5-prime linked dimer became the most reactive, yielding chains of the order of 60 monomer-unit lengths. Remarkable self-organization properties are noted for the 5-prime-5-prime dimer of pdAp.

  16. Oligomerization of deoxynucleoside-bisphosphate dimers: template and linkage specificity

    NASA Technical Reports Server (NTRS)

    Visscher, J.; van der Woerd, R.; Bakker, C. G.; Schwartz, A. W.; Orgel, L. E. (Principal Investigator)

    1989-01-01

    Evidence is presented that a poly(U) template selectively favors the oligomerization of the activated, 3'-5' pyrophosphate-linked dimer pdAppdAp, in comparison with the 3'-3' and 5'-5' linked dimers. In the absence of poly(U), the 5'-5' linked dimer is the most reactive, and chains are formed which are more than 60 monomer units in length.

  17. Mutation of the highly conserved Ser-40 of the HIV-1 p6 gag protein to Phe causes the formation of a hydrophobic patch, enhances membrane association, and polyubiquitination of Gag.

    PubMed

    Hahn, Friedrich; Setz, Christian; Friedrich, Melanie; Rauch, Pia; Solbak, Sara Marie; Frøystein, Nils Age; Henklein, Petra; Votteler, Jörg; Fossen, Torgils; Schubert, Ulrich

    2014-10-02

    The HIV-1 p6 Gag protein contains two late assembly (l-) domains that recruit proteins of the endosomal sorting complex required for transport (ESCRT) pathway to mediate membrane fission between the nascent virion and the cell membrane. It was recently demonstrated that mutation of the highly conserved Ser-40 to Phe (S40F) disturbs CA-SP1 processing, virus morphogenesis, and infectivity. It also causes the formation of filopodia-like structures, while virus release remains unaffected. Here, we show that the mutation S40F, but not the conservative mutation to Asp (S40D) or Asn (S40N), augments membrane association, K48-linked polyubiquitination, entry into the 26S proteasome, and, consequently, enhances MHC-I antigen presentation of Gag derived epitopes. Nuclear magnetic resonance (NMR) structure analyses revealed that the newly introduced Phe-40, together with Tyr-36, causes the formation of a hydrophobic patch at the C-terminal α-helix of p6, providing a molecular rationale for the enhanced membrane association of Gag observed in vitro and in HIV-1 expressing cells. The extended exposure of the S40F mutant to unidentified membrane-resident ubiquitin E3-ligases might trigger the polyubiquitination of Gag. The cumulative data support a previous model of a so far undefined property of p6, which, in addition to MA, acts as membrane targeting domain of Gag.

  18. Mutation of the Highly Conserved Ser-40 of the HIV-1 p6 Gag Protein to Phe Causes the Formation of a Hydrophobic Patch, Enhances Membrane Association, and Polyubiquitination of Gag

    PubMed Central

    Hahn, Friedrich; Setz, Christian; Friedrich, Melanie; Rauch, Pia; Solbak, Sara Marie; Frøystein, Nils Åge; Henklein, Petra; Votteler, Jörg; Fossen, Torgils; Schubert, Ulrich

    2014-01-01

    The HIV-1 p6 Gag protein contains two late assembly (l-) domains that recruit proteins of the endosomal sorting complex required for transport (ESCRT) pathway to mediate membrane fission between the nascent virion and the cell membrane. It was recently demonstrated that mutation of the highly conserved Ser-40 to Phe (S40F) disturbs CA-SP1 processing, virus morphogenesis, and infectivity. It also causes the formation of filopodia-like structures, while virus release remains unaffected. Here, we show that the mutation S40F, but not the conservative mutation to Asp (S40D) or Asn (S40N), augments membrane association, K48-linked polyubiquitination, entry into the 26S proteasome, and, consequently, enhances MHC-I antigen presentation of Gag derived epitopes. Nuclear magnetic resonance (NMR) structure analyses revealed that the newly introduced Phe-40, together with Tyr-36, causes the formation of a hydrophobic patch at the C-terminal α-helix of p6, providing a molecular rationale for the enhanced membrane association of Gag observed in vitro and in HIV-1 expressing cells. The extended exposure of the S40F mutant to unidentified membrane-resident ubiquitin E3-ligases might trigger the polyubiquitination of Gag. The cumulative data support a previous model of a so far undefined property of p6, which, in addition to MA, acts as membrane targeting domain of Gag. PMID:25279819

  19. [Antinuclear antibodies].

    PubMed

    Cabiedes, Javier; Núñez-Álvarez, Carlos A

    2010-01-01

    Anti-nuclear antibodies (ANA) are immunoglobulin directed against autologous cell nuclear and cytoplasmic components. Besides the autoimmune ANA there are other ANA that can be detected in circulation, like natural and infectious ANA. Because of its high sensibility, detection of the ANA must be done by indirect immunofluorescence (IIF) as screening test and all of those positive samples are convenient to confirm its specificity by ELISA, western blot or other techniques. Positive ANA detected by IIF must be evaluated taking in to account the pattern and titer. The following recommended step is the specificity characterization (reactivity against extractable nuclear antigens [ENA], dsDNA, etc.) which is useful for the diagnosis and follow up of patients with autoimmune diseases, and by such reasoning, its detection must be performed in an orderly and reasonable way using guides or strategies focused to the good use and interpretation of the autoantibodies. The objective of this review is to present a compilation of the literature and our experience in the detection and study of the ANA.

  20. Selection of antibodies from synthetic antibody libraries.

    PubMed

    Harel Inbar, Noa; Benhar, Itai

    2012-10-15

    More than 2 dozen years had passed since the field of antibody engineering was established, with the first reports of bacterial [1-3] and mammalian cells [4] expression of recombinant antibody fragments, and in that time a lot of effort was dedicated to the development of efficient technological means, intended to assist in the creation of therapeutic monoclonal antibodies (mAbs). Research focus was given to two intertwined technological aspects: the selection platform and the recombinant antibody repertoires. In accordance with these areas of interest, it is the goal of this chapter to describe the various selection tools and antibody libraries existing, with emphasis on the later, and their applications. This chapter gives a far from exhaustive, subjective "historic account" of the field, describing the selection platforms, the different formats of antibody repertoires and the applications of both for selecting recombinant antibodies. Several excellent books provide detailed protocols for constructing antibody libraries and selecting antibodies from those libraries [5-13]. Such books may guide a newcomer to the field in the fine details of antibody engineering. We would like to offer advice to the novice: although seemingly simple, effective library construction and antibody isolation provide best benefits in the hands of professionals. It is an art as much as it is science.

  1. Regulation of activation-induced deaminase stability and antibody gene diversification by Hsp90

    PubMed Central

    Orthwein, Alexandre; Patenaude, Anne-Marie; Affar, El Bachir; Lamarre, Alain; Young, Jason C.

    2010-01-01

    Activation-induced deaminase (AID) is the mutator enzyme that initiates somatic hypermutation and isotype switching of the antibody genes in B lymphocytes. Undesired byproducts of AID function are oncogenic mutations. AID expression levels seem to correlate with the extent of its physiological and pathological functions. In this study, we identify AID as a novel Hsp90 (heat shock protein 90 kD) client. We find that cytoplasmic AID is in a dynamic equilibrium regulated by Hsp90. Hsp90 stabilizes cytoplasmic AID, as specific Hsp90 inhibition leads to cytoplasmic polyubiquitination and proteasomal degradation of AID. Consequently, Hsp90 inhibition results in a proportional reduction in antibody gene diversification and off-target mutation. This evolutionarily conserved regulatory mechanism determines the functional steady-state levels of AID in normal B cells and B cell lymphoma lines. Thus, Hsp90 assists AID-mediated antibody diversification by stabilizing AID. Hsp90 inhibition provides the first pharmacological means to down-regulate AID expression and activity, which could be relevant for therapy of some lymphomas and leukemias. PMID:21041454

  2. Antibodies and antibody-derived analytical biosensors

    PubMed Central

    Sharma, Shikha; Byrne, Hannah

    2016-01-01

    The rapid diagnosis of many diseases and timely initiation of appropriate treatment are critical determinants that promote optimal clinical outcomes and general public health. Biosensors are now being applied for rapid diagnostics due to their capacity for point-of-care use with minimum need for operator input. Antibody-based biosensors or immunosensors have revolutionized diagnostics for the detection of a plethora of analytes such as disease markers, food and environmental contaminants, biological warfare agents and illicit drugs. Antibodies are ideal biorecognition elements that provide sensors with high specificity and sensitivity. This review describes monoclonal and recombinant antibodies and different immobilization approaches crucial for antibody utilization in biosensors. Examples of applications of a variety of antibody-based sensor formats are also described. PMID:27365031

  3. Serum herpes simplex antibodies

    MedlinePlus

    ... page: //medlineplus.gov/ency/article/003352.htm Serum herpes simplex antibodies To use the sharing features on this page, please enable JavaScript. Serum herpes simplex antibodies is a blood test that looks for ...

  4. Platelet associated antibodies

    MedlinePlus

    ... medlineplus.gov/ency/article/003552.htm Platelet-associated antibodies blood test To use the sharing features on ... JavaScript. This blood test shows if you have antibodies against platelets in your blood. Platelets are a ...

  5. RBC Antibody Screen

    MedlinePlus

    ... Cell Antibody Screen Related tests: Direct Antiglobulin Test ; Blood Typing ; RBC Antibody Identification ; Type and Screen; Crossmatch All content on Lab Tests Online has been reviewed and approved by our Editorial Review Board . At a ... screen is used to screen an individual's blood for antibodies directed against red blood cell (RBC) ...

  6. Modeling Antibody Diversity.

    ERIC Educational Resources Information Center

    Baker, William P.; Moore, Cathy Ronstadt

    1998-01-01

    Understanding antibody structure and function is difficult for many students. The rearrangement of constant and variable regions during antibody differentiation can be effectively simulated using a paper model. Describes a hands-on laboratory exercise which allows students to model antibody diversity using readily available resources. (PVD)

  7. Modeling Antibody Diversity.

    ERIC Educational Resources Information Center

    Baker, William P.; Moore, Cathy Ronstadt

    1998-01-01

    Understanding antibody structure and function is difficult for many students. The rearrangement of constant and variable regions during antibody differentiation can be effectively simulated using a paper model. Describes a hands-on laboratory exercise which allows students to model antibody diversity using readily available resources. (PVD)

  8. Antibody Therapeutics in Oncology

    PubMed Central

    Wold, Erik D; Smider, Vaughn V; Felding, Brunhilde H

    2016-01-01

    One of the newer classes of targeted cancer therapeutics is monoclonal antibodies. Monoclonal antibody therapeutics are a successful and rapidly expanding drug class due to their high specificity, activity, favourable pharmacokinetics, and standardized manufacturing processes. Antibodies are capable of recruiting the immune system to attack cancer cells through complement-dependent cytotoxicity or antibody dependent cellular cytotoxicity. In an ideal scenario the initial tumor cell destruction induced by administration of a therapeutic antibody can result in uptake of tumor associated antigens by antigen-presenting cells, establishing a prolonged memory effect. Mechanisms of direct tumor cell killing by antibodies include antibody recognition of cell surface bound enzymes to neutralize enzyme activity and signaling, or induction of receptor agonist or antagonist activity. Both approaches result in cellular apoptosis. In another and very direct approach, antibodies are used to deliver drugs to target cells and cause cell death. Such antibody drug conjugates (ADCs) direct cytotoxic compounds to tumor cells, after selective binding to cell surface antigens, internalization, and intracellular drug release. Efficacy and safety of ADCs for cancer therapy has recently been greatly advanced based on innovative approaches for site-specific drug conjugation to the antibody structure. This technology enabled rational optimization of function and pharmacokinetics of the resulting conjugates, and is now beginning to yield therapeutics with defined, uniform molecular characteristics, and unprecedented promise to advance cancer treatment. PMID:27081677

  9. Antibodies in Transplantation

    PubMed Central

    Platt, Jeffrey L.

    2011-01-01

    Transplantation of cells, tissues, and organs from one individual to another can incite the production of antibodies specific for foreign antigens, especially major histocompatibility antigens, in the graft. Antibodies specific for a graft provide an index of immunity and a potential trigger for injury and rejection. However, the index of immunity can sometimes miss antibody-mediated rejection and besides causing injury the antibodies against a graft can also protect a graft from injury by blocking immune recognition, called enhancement, regulating activation of complement, and inducing changes in the graft that resist damage. Reviewed here are potential limitations in the use of antibodies as an index of immunity and the ways antibodies cause and/or prevent injury. PMID:20807473

  10. Demystified...recombinant antibodies.

    PubMed

    Smith, K A; Nelson, P N; Warren, P; Astley, S J; Murray, P G; Greenman, J

    2004-09-01

    Recombinant antibodies are important tools for biomedical research and are increasingly being used as clinical diagnostic/therapeutic reagents. In this article, a background to humanized antibodies is given, together with details of the generation of antibody fragments--for example, single chain Fv fragments. Phage antibody fragments are fast becoming popular and can be generated by simple established methods of affinity enrichment from libraries derived from immune cells. Phage display methodology can also be used for the affinity enrichment of existing antibody fragments to provide a reagent with a higher affinity. Here, phage antibodies are demystified to provide a greater understanding of the potential of these reagents and to engage clinicians and biomedical scientists alike to think about potential applications in pathology and clinical settings.

  11. SARS Coronavirus Papain-Like Protease Inhibits the TLR7 Signaling Pathway through Removing Lys63-Linked Polyubiquitination of TRAF3 and TRAF6.

    PubMed

    Li, Shih-Wen; Wang, Ching-Ying; Jou, Yu-Jen; Huang, Su-Hua; Hsiao, Li-Hsin; Wan, Lei; Lin, Ying-Ju; Kung, Szu-Hao; Lin, Cheng-Wen

    2016-05-05

    Severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLPro) reportedly inhibits the production of type I interferons (IFNs) and pro-inflammatory cytokines in Toll-like receptor 3 (TLR3) and retinoic acid-inducible gene 1 (RIG-I) pathways. The study investigated the inhibitory effect and its antagonistic mechanism of SARS-CoV PLPro on TLR7-mediated cytokine production. TLR7 agonist (imiquimod (IMQ)) concentration-dependently induced activation of ISRE-, NF-κB- and AP-1-luciferase reporters, as well as the production of IFN-α, IFN-β, TNF-α, IL-6 and IL-8 in human promonocyte cells. However, SARS-CoV PLPro significantly inhibited IMQ-induced cytokine production through suppressing the activation of transcription factors IRF-3, NF-κB and AP-1. Western blot analysis with anti-Lys48 and anti-Lys63 ubiquitin antibodies indicated the SARS-CoV PLPro removed Lys63-linked ubiquitin chains of TRAF3 and TRAF6, but not Lys48-linked ubiquitin chains in un-treated and treated cells. The decrease in the activated state of TRAF3 and TRAF6 correlated with the inactivation of TBK1 in response to IMQ by PLPro. The results revealed that the antagonism of SARS-CoV PLPro on TLR7-mediated innate immunity was associated with the negative regulation of TRAF3/6-TBK1-IRF3/NF-κB/AP1 signals.

  12. Tumor Necrosis Factor (TNF) Receptor-associated Factor 7 Is Required for TNFα-induced Jun NH2-terminal Kinase Activation and Promotes Cell Death by Regulating Polyubiquitination and Lysosomal Degradation of c-FLIP Protein*

    PubMed Central

    Scudiero, Ivan; Zotti, Tiziana; Ferravante, Angela; Vessichelli, Mariangela; Reale, Carla; Masone, Maria C.; Leonardi, Antonio; Vito, Pasquale; Stilo, Romania

    2012-01-01

    The pro-inflammatory cytokine tumor necrosis factor (TNF) α signals both cell survival and death. The biological outcome of TNFα treatment is determined by the balance between survival factors and Jun NH2-terminal kinase (JNK) signaling, which promotes cell death. Here, we show that TRAF7, the most recently identified member of the TNF receptor-associated factors (TRAFs) family of proteins, is essential for activation of JNK following TNFα stimulation. We also show that TRAF6 and TRAF7 promote unconventional polyubiquitination of the anti-apoptotic protein c-FLIPL and demonstrate that degradation of c-FLIPL also occurs through a lysosomal pathway. RNA interference-mediated depletion of TRAF7 correlates with increased c-FLIPL expression level, which, in turn, results in resistance to TNFα cytotoxicity. Collectively, our results indicate an important role for TRAF7 in the activation of JNK following TNFα stimulation and clearly point to an involvement of this protein in regulating the turnover of c-FLIP and, consequently, cell death. PMID:22219201

  13. TRAF7 protein promotes Lys-29-linked polyubiquitination of IkappaB kinase (IKKgamma)/NF-kappaB essential modulator (NEMO) and p65/RelA protein and represses NF-kappaB activation.

    PubMed

    Zotti, Tiziana; Uva, Antonio; Ferravante, Angela; Vessichelli, Mariangela; Scudiero, Ivan; Ceccarelli, Michele; Vito, Pasquale; Stilo, Romania

    2011-07-01

    Tumor necrosis factor receptor-associated factor (TRAF) proteins are cytoplasmic regulatory molecules that function as signal transducers for receptors involved in both innate and adaptive humoral immune responses. In this study, we show that TRAF7, the unique noncanonical member of the TRAF family, physically associates with IκB kinase/NF-κB essential modulator (NEMO) and with the RelA/p65 (p65) member of the NF-κB transcription factor family. TRAF7 promotes Lys-29-linked polyubiquitination of NEMO and p65 that results in lysosomal degradation of both proteins and altered activation. TRAF7 also influences p65 nuclear distribution. Microarray expression data are consistent with an inhibitory role for TRAF7 on NF-κB and a positive control of AP-1 transcription factor. Finally, functional data indicate that TRAF7 promotes cell death. Thus, this study identifies TRAF7 as a NEMO- and p65-interacting molecule and brings important information on the ubiquitination events that control NF-κB transcriptional activity.

  14. Tumor necrosis factor (TNF) receptor-associated factor 7 is required for TNFα-induced Jun NH2-terminal kinase activation and promotes cell death by regulating polyubiquitination and lysosomal degradation of c-FLIP protein.

    PubMed

    Scudiero, Ivan; Zotti, Tiziana; Ferravante, Angela; Vessichelli, Mariangela; Reale, Carla; Masone, Maria C; Leonardi, Antonio; Vito, Pasquale; Stilo, Romania

    2012-02-17

    The pro-inflammatory cytokine tumor necrosis factor (TNF) α signals both cell survival and death. The biological outcome of TNFα treatment is determined by the balance between survival factors and Jun NH(2)-terminal kinase (JNK) signaling, which promotes cell death. Here, we show that TRAF7, the most recently identified member of the TNF receptor-associated factors (TRAFs) family of proteins, is essential for activation of JNK following TNFα stimulation. We also show that TRAF6 and TRAF7 promote unconventional polyubiquitination of the anti-apoptotic protein c-FLIP(L) and demonstrate that degradation of c-FLIP(L) also occurs through a lysosomal pathway. RNA interference-mediated depletion of TRAF7 correlates with increased c-FLIP(L) expression level, which, in turn, results in resistance to TNFα cytotoxicity. Collectively, our results indicate an important role for TRAF7 in the activation of JNK following TNFα stimulation and clearly point to an involvement of this protein in regulating the turnover of c-FLIP and, consequently, cell death.

  15. TRAF7 Protein Promotes Lys-29-linked Polyubiquitination of IκB Kinase (IKKγ)/NF-κB Essential Modulator (NEMO) and p65/RelA Protein and Represses NF-κB Activation

    PubMed Central

    Zotti, Tiziana; Uva, Antonio; Ferravante, Angela; Vessichelli, Mariangela; Scudiero, Ivan; Ceccarelli, Michele; Vito, Pasquale; Stilo, Romania

    2011-01-01

    Tumor necrosis factor receptor-associated factor (TRAF) proteins are cytoplasmic regulatory molecules that function as signal transducers for receptors involved in both innate and adaptive humoral immune responses. In this study, we show that TRAF7, the unique noncanonical member of the TRAF family, physically associates with IκB kinase/NF-κB essential modulator (NEMO) and with the RelA/p65 (p65) member of the NF-κB transcription factor family. TRAF7 promotes Lys-29-linked polyubiquitination of NEMO and p65 that results in lysosomal degradation of both proteins and altered activation. TRAF7 also influences p65 nuclear distribution. Microarray expression data are consistent with an inhibitory role for TRAF7 on NF-κB and a positive control of AP-1 transcription factor. Finally, functional data indicate that TRAF7 promotes cell death. Thus, this study identifies TRAF7 as a NEMO- and p65-interacting molecule and brings important information on the ubiquitination events that control NF-κB transcriptional activity. PMID:21518757

  16. Expression of recombinant antibodies.

    PubMed

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with "human-like" post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

  17. Recombinant renewable polyclonal antibodies.

    PubMed

    Ferrara, Fortunato; D'Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew R M

    2015-01-01

    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.

  18. Recombinant renewable polyclonal antibodies

    PubMed Central

    Ferrara, Fortunato; D’Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew RM

    2015-01-01

    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products. PMID:25530082

  19. Therapeutic Recombinant Monoclonal Antibodies

    ERIC Educational Resources Information Center

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  20. Production Of Human Antibodies

    NASA Technical Reports Server (NTRS)

    Sammons, David W.; Neil, Garry A.

    1993-01-01

    Process for making human monoclonal antibodies based on combination of techniques. Antibodies made active against specific antigen. Process involves in vivo immunization of human B lymphocyte cells in mice. B cells of interest enriched in vitro before fusion. Method potentially applicable to any antigen. Does not rely on use of Epstein-Barr virus at any step. Human lymphocytes taken from any source.

  1. Affinity purification of antibodies

    USDA-ARS?s Scientific Manuscript database

    Antibodies are provided in a variety of formats that includes antiserum, hybridoma culture supernatant or ascites. They can all be used successfully in crude form for the detection of target antigens by immunoassay. However, it is advantageous to use purified antibody in defined quantity to facil...

  2. Production Of Human Antibodies

    NASA Technical Reports Server (NTRS)

    Sammons, David W.; Neil, Garry A.

    1993-01-01

    Process for making human monoclonal antibodies based on combination of techniques. Antibodies made active against specific antigen. Process involves in vivo immunization of human B lymphocyte cells in mice. B cells of interest enriched in vitro before fusion. Method potentially applicable to any antigen. Does not rely on use of Epstein-Barr virus at any step. Human lymphocytes taken from any source.

  3. Expression of Recombinant Antibodies

    PubMed Central

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications. PMID:23908655

  4. Therapeutic Recombinant Monoclonal Antibodies

    ERIC Educational Resources Information Center

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  5. Antibodies for biodefense

    PubMed Central

    Froude, Jeffrey W; Stiles, Bradley; Pelat, Thibaut

    2011-01-01

    Potential bioweapons are biological agents (bacteria, viruses and toxins) at risk of intentional dissemination. Biodefense, defined as development of therapeutics and vaccines against these agents, has seen an increase, particularly in the US, following the 2001 anthrax attack. This review focuses on recombinant antibodies and polyclonal antibodies for biodefense that have been accepted for clinical use. These antibodies aim to protect against primary potential bioweapons or category A agents as defined by the Centers for Disease Control and Prevention (Bacillus anthracis, Yersinia pestis, Francisella tularensis, botulinum neurotoxins, smallpox virus and certain others causing viral hemorrhagic fevers) and certain category B agents. Potential for prophylactic use is presented, as well as frequent use of oligoclonal antibodies or synergistic effect with other molecules. Capacities and limitations of antibodies for use in biodefense are discussed, and are generally applicable to the field of infectious diseases. PMID:22123065

  6. Anti-insulin antibody test

    MedlinePlus

    Insulin antibodies - serum; Insulin Ab test; Insulin resistance - insulin antibodies; Diabetes - insulin antibodies ... You appear to have an allergic response to insulin Insulin no longer seems to control your diabetes

  7. Selection of Recombinant Human Antibodies.

    PubMed

    Tomszak, Florian; Weber, Susanne; Zantow, Jonas; Schirrmann, Thomas; Hust, Michael; Frenzel, André

    2016-01-01

    Since the development of therapeutic antibodies the demand of recombinant human antibodies is steadily increasing. Traditionally, therapeutic antibodies were generated by immunization of rat or mice, the generation of hybridoma clones, cloning of the antibody genes and subsequent humanization and engineering of the lead candidates. In the last few years, techniques were developed that use transgenic animals with a human antibody gene repertoire. Here, modern recombinant DNA technologies can be combined with well established immunization and hybridoma technologies to generate already affinity maturated human antibodies. An alternative are in vitro technologies which enabled the generation of fully human antibodies from antibody gene libraries that even exceed the human antibody repertoire. Specific antibodies can be isolated from these libraries in a very short time and therefore reduce the development time of an antibody drug at a very early stage.In this review, we describe different technologies that are currently used for the in vitro and in vivo generation of human antibodies.

  8. Red Blood Cell Antibody Identification

    MedlinePlus

    ... name: Red Blood Cell Antibody Identification Related tests: Direct Antiglobulin Test ; RBC Antibody Screen ; Blood Typing ; Type ... a positive RBC antibody screen or a positive direct antiglobulin test (DAT) . It is used to identify ...

  9. Stereospecific antibodies to propranolol.

    PubMed

    Wang, L; Chorev, M; Feingers, J; Levitzki, A; Inbar, M

    1986-04-21

    The beta-adrenergic antagonist propranolol was activated through its side chain, coupled to bovine serum albumin, and injected into BALB/c mice. After fusion of the splenocytes from these immunized mice with the NS-1 myeloma cell line, two hybridomas, producing monoclonal anti-propranolol antibodies, were isolated. Clone P-49 was monospecific for propranolol, with a significant preference for the 1-stereoisomer, as compared to the d form. On the other hand, clone P-28 cross-reacted with alprenolol as well as some other beta-antagonists. Both classes of antibodies competed with A431 epidermoid carcinoma beta 2-adrenoceptors for the binding of [3H]propranolol. When ascites cells from clone P-28 were fixed with glutaraldehyde, the anti-propranolol monoclonal antibody became cell bound. These cell-bound P-28 antibodies bind propranolol and other beta-adrenergic ligands with a similar ranking order to the soluble monoclonal antibody. The cell-bound antibody displayed a 5-fold higher affinity towards 1-propranolol than the soluble monoclonal antibody. The practical implications of these findings are discussed.

  10. STUDIES ON ANTIBODY PRODUCTION

    PubMed Central

    White, Robert G.; Coons, Albert H.; Connolly, Jeanne M.

    1955-01-01

    After subcutaneous injection of hen's ovalbumin or diphtheria toxoid precipitated with aluminum phosphate, the production of antibody, as judged by the presence in the tissues of antibody-containing cells, proceeds partly within the regional lymphatic glands and partly in the granulation tissue surrounding the nodule which develops at the site of injection. The first production of antibody takes place in the regional lymphatic gland and antibody production in the local granuloma becomes apparent only from 14 days onwards (rabbit). Antibody-containing plasma cells were demonstrated in the local granuloma up to 7 weeks. Antibody-containing cells in the regional lymphatic glands reach maximum numbers at 2 weeks following injection and decrease thereafter to few cells at 5 weeks. The adjuvant effect of the aluminum phosphate is interpreted as due partly to the delay in absorption of antigen from the local site of its injection which results in prolongation of stimulation of cells within the regional lymphatic glands, and partly to the production of a local granuloma which contains antibody-producing plasma cells. PMID:14392242

  11. Therapeutic antibody technology 97.

    PubMed

    Larrick, J W; Gavilondo, J

    1998-01-01

    Almost 200 antibody aficionados attended the Therapeutic Antibody Technology 97 meeting, held September 21-24, 1997 at the Holiday Inn, Union Square in the heart of San Francisco, CA. The meeting was sponsored by the Palo Alto Institute of Molecular Medicine and organized by James W. Larrick (PAIMM) and Dennis R. Burton (Scripps Research Institute). The meeting featured excellent discussions on many interesting talks and a number of poster presentations. It is likely that another meeting will be organized in 2 years, however in the meantime, an effort is underway to organize a 'Virtual Antibody Society' to be set up on the web server at Scripps Research Institute in La Jolla, CA (Questions and comments on this project can be sent to: Jwlarrick@aol.com or Burton@scripps.edu). Richard Lerner (Scripps) gave the keynote address on 'Catalytic Antibodies', describing recent work with Carlos Barbas on so-called reactive immunization to generate a high activity aldolase catalytic antibody. This antibody, soon to be described in an article in Science, is the first commercially available catalytic antibody.

  12. NMDA receptor antibodies

    PubMed Central

    Ramberger, Melanie; Bsteh, Gabriel; Schanda, Kathrin; Höftberger, Romana; Rostásy, Kevin; Baumann, Matthias; Aboulenein-Djamshidian, Fahmy; Lutterotti, Andreas; Deisenhammer, Florian; Berger, Thomas

    2015-01-01

    Objectives: To analyze the frequency of NMDA receptor (NMDAR) antibodies in patients with various inflammatory demyelinating diseases of the CNS and to determine their clinical correlates. Methods: Retrospective case-control study from 2005 to 2014 with the detection of serum IgG antibodies to NMDAR, aquaporin-4, and myelin oligodendrocyte glycoprotein by recombinant live cell-based immunofluorescence assays. Fifty-one patients with acute disseminated encephalomyelitis, 41 with neuromyelitis optica spectrum disorders, 34 with clinically isolated syndrome, and 89 with multiple sclerosis (MS) were included. Due to a known association of NMDAR antibodies with seizures and behavioral symptoms, patients with those clinical manifestations were preferentially included and are therefore overrepresented in our cohort. Nine patients with NMDAR encephalitis, 94 patients with other neurologic diseases, and 48 healthy individuals were used as controls. Results: NMDAR antibodies were found in all 9 patients with NMDAR encephalitis but in only 1 of 215 (0.5%) patients with inflammatory demyelination and in none of the controls. This patient had relapsing-remitting MS with NMDAR antibodies present at disease onset, with an increase in NMDAR antibody titer with the onset of psychiatric symptoms and cognitive deficits. Conclusion: In demyelinating disorders, NMDAR antibodies are uncommon, even in those with symptoms seen in NMDAR encephalitis. PMID:26309901

  13. Antibody discovery: sourcing of monoclonal antibody variable domains.

    PubMed

    Strohl, William R

    2014-03-01

    Historically, antibody variable domains for therapeutic antibodies have been sourced primarily from the mouse IgG repertoire, and typically either chimerized or humanized. More recently, human antibodies from transgenic mice producing human IgG, phage display libraries, and directly from human B lymphocytes have been used more broadly as sources of antibody variable domains for therapeutic antibodies. Of the total 36 antibodies approved by major maket regulatory agencies, the variable domain sequences of 26 originate from the mouse. Of these, four are marketed as murine antibodies (of which one is a mouse-rat hybrid IgG antibody), six are mouse-human chimeric antibodies, and 16 are humanized. Ten marketed antibodies have originated from human antibody genes, three isolated from phage libraries of human antibody genes and seven from transgenic mice producing human antibodies. Five antibodies currently in clinical trials have been sourced from camelids, as well as two from non-human primates, one from rat, and one from rabbit. Additional sources of antibody variable domains that may soon find their way into the clinic are potential antibodies from sharks and chickens. Finally, the various methods for retrieval of antibodies from humans, mouse and other sources, including various display technologies and amplification directly from B cells, are described.

  14. Monoclonal antibody "gold rush".

    PubMed

    Maggon, Krishan

    2007-01-01

    The market, sales and regulatory approval of new human medicines, during the past few years, indicates increasing number and share of new biologics and emergence of new multibillion dollar molecules. The global sale of monoclonal antibodies in 2006 were $20.6 billion. Remicade had annual sales gain of $1 billion during the past 3 years and five brands had similar increase in 2006. Rituxan with 2006 sales of $4.7 billion was the best selling monoclonal antibody and biological product and the 6th among the top selling medicinal brand. It may be the first biologic and monoclonal antibody to reach $10 billion annual sales in the near future. The strong demand from cancer and arthritis patients has surpassed almost all commercial market research reports and sales forecast. Seven monoclonal antibody brands in 2006 had sales exceeding $1 billion. Humanized or fully human monoclonal antibodies with low immunogenicity, enhanced antigen binding and reduced cellular toxicity provide better clinical efficacy. The higher technical and clinical success rate, overcoming of technical hurdles in large scale manufacturing, low cost of market entry and IND filing, use of fully human and humanized monoclonal antibodies has attracted funds and resources towards R&D. Review of industry research pipeline and sales data during the past 3 years indicate a real paradigm shift in industrial R&D from pharmaceutical to biologics and monoclonal antibodies. The antibody bandwagon has been joined by 200 companies with hundreds of new projects and targets and has attracted billions of dollars in R&D investment, acquisitions and licensing deals leading to the current Monoclonal Antibody Gold Rush.

  15. Antiphospholipid antibody syndrome.

    PubMed

    Kutteh, William H; Hinote, Candace D

    2014-03-01

    Antiphospholipid antibodies (aPLs) are acquired antibodies directed against negatively charged phospholipids. Obstetric antiphospholipid antibody syndrome (APS) is diagnosed in the presence of certain clinical features in conjunction with positive laboratory findings. Obstetric APS is one of the most commonly identified causes of recurrent pregnancy loss. Thus, obstetric APS is distinguished from APS in other organ systems where the most common manifestation is thrombosis. Several pathophysiologic mechanisms of action of aPLs have been described. This article discusses the diagnostic and obstetric challenges of obstetric APS, proposed pathophysiologic mechanisms of APS during pregnancy, and the management of women during and after pregnancy.

  16. Anti-cartilage antibody.

    PubMed Central

    Greenbury, C L; Skingle, J

    1979-01-01

    Antibody to cartilage has been demonstrated by indirect immunofluorescence on rat trachea in the serum of about 3% of 1126 patients with rheumatoid arthritis. Titres ranged from 1:20 to 1:640. The antibody was not found in 284 patients with primary or secondary osteoarthritis or in 1825 blood donors, nor, with the exception of two weak reactors, in 1314 paraplegic patients. In most cases the antibody appears to be specific for native type II collagen. Using this as an antigen in a haemagglutination test 94% of anti-cartilage sera were positive, whereas among 100 rheumatoid control sera there were only three weak positives. More than 80% of patients with antibody had some erosion of articular cartilage, but there was no correlation with age, sex, duration of disease, nor any recognisable clinical event or change. Images Fig. 1 PMID:389957

  17. Heterogeneity of monoclonal antibodies.

    PubMed

    Liu, Hongcheng; Gaza-Bulseco, Georgeen; Faldu, Dinesh; Chumsae, Chris; Sun, Joanne

    2008-07-01

    Heterogeneity of monoclonal antibodies is common due to the various modifications introduced over the lifespan of the molecules from the point of synthesis to the point of complete clearance from the subjects. The vast number of modifications presents great challenge to the thorough characterization of the molecules. This article reviews the current knowledge of enzymatic and nonenzymatic modifications of monoclonal antibodies including the common ones such as incomplete disulfide bond formation, glycosylation, N-terminal pyroglutamine cyclization, C-terminal lysine processing, deamidation, isomerization, and oxidation, and less common ones such as modification of the N-terminal amino acids by maleuric acid and amidation of the C-terminal amino acid. In addition, noncovalent associations with other molecules, conformational diversity and aggregation of monoclonal antibodies are also discussed. Through a complete understanding of the heterogeneity of monoclonal antibodies, strategies can be employed to better identify the potential modifications and thoroughly characterize the molecules.

  18. HIV Antibody Test

    MedlinePlus

    ... AACC products and services. Advertising & Sponsorship: Policy | Opportunities HIV Antibody and HIV Antigen (p24) Share this page: Was this page helpful? Also known as: HIV Screening Tests; AIDS Test; AIDS Screen; HIV Serology; ...

  19. Mining human antibody repertoires

    PubMed Central

    2010-01-01

    Human monoclonal antibodies (mAbs) have become drugs of choice for the management of an increasing number of human diseases. Human antibody repertoires provide a rich source for human mAbs. Here we review the characteristics of natural and non-natural human antibody repertoires and their mining with non-combinatorial and combinatorial strategies. In particular, we discuss the selection of human mAbs from naïve, immune, transgenic and synthetic human antibody repertoires using methods based on hybridoma technology, clonal expansion of peripheral B cells, single-cell PCR, phage display, yeast display and mammalian cell display. Our reliance on different strategies is shifting as we gain experience and refine methods to the efficient generation of human mAbs with superior pharmacokinetic and pharmacodynamic properties. PMID:20505349

  20. Anti-sulfotyrosine antibodies

    DOEpatents

    Bertozzi, Carolyn R [Berkeley, CA; Kehoe, John [Saint Davids, PA; Bradbury, Andrew M [Santa Fe, NM

    2009-09-15

    The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

  1. Antibody tumor penetration

    PubMed Central

    Thurber, Greg M.; Schmidt, Michael M.; Wittrup, K. Dane

    2009-01-01

    Antibodies have proven to be effective agents in cancer imaging and therapy. One of the major challenges still facing the field is the heterogeneous distribution of these agents in tumors when administered systemically. Large regions of untargeted cells can therefore escape therapy and potentially select for more resistant cells. We present here a summary of theoretical and experimental approaches to analyze and improve antibody penetration in tumor tissue. PMID:18541331

  2. Monoclonal antibodies and cancer therapy

    SciTech Connect

    Reisfeld, R.A.; Sell, S.

    1985-01-01

    These proceedings collect papers on the subject of monoclonal antibodies. Topics include: Monoclonal antibody, biochemical effects and cancer therapeutic potential of tunicamycin, use of monoclonal antibodies for detection of lymph node metastases, active specific immunotherapy, and applications of monoclonal antibodies to investigations of growth factors.

  3. [New antibodies in cancer treatment].

    PubMed

    Pestalozzi, B C; Knuth, A

    2004-09-22

    Since the development of hybridoma technology in 1975 monoclonal antibodies with pre-defined specificity can be produced. Only twenty years later did it become possible to make therapeutic use of monoclonal antibodies in oncology. To this end it was necessary to attach the antigen-binding site of a mouse antibody onto the scaffold of a human antibody molecule. Such chimeric or "humanized" antibodies may be used in passive immunotherapy without eliciting an immune response. Rituximab and trastuzumab are such humanized antibodies. They are used today routinely in the treatment of malignant lymphoma and breast cancer, respectively. These antibodies are usually used in combination with conventional cytostatic anticancer drugs.

  4. Antibody decoration of neurofilaments

    PubMed Central

    1981-01-01

    We have decorated neurofilaments with antibodies against three polypeptides (designated here as H [mol wt = 195,000], 45[mol wt = 145,000], and 46[mol wt = 73,000]) in an effort to understand the arrangement of these polypeptides within neurofilaments. The three polypeptides were purified and antibodies were raised against each. The cross-reactivity of the antibodies suggested that each polypeptide contains both shared and unique antigenic determinants. The differential reactivities of each antibody preparation were enhanced by adsorption with the two heterologous polypeptides, and the resulting preparations were used to decorate purified neurofilaments, which were then negatively stained and examined in an electron microscope. The appearance of the antibody-decorated structures led to the following conclusions: All three polypeptides are physically associated with the same neurofilament. However, the disposition of H and 46 within a filament is different; 46 antigens appear to be associated with a "central core" of the filament, whereas H antigens compose a structure more loosely and peripherally attached to the central core and periodically arranged along its axis. The antibody-decorated H- containing structure assumes variable configurations; in some cases it appears asa bridge connecting two filaments; in other cases it appears as a helix wrapping the central core with a period of approximately 1,000 A and an apparent unit length of approximately 1.5 periods. These configurations suggest several functional implications, including the possibility that H is a component of the cross-bridges observed between filaments in situ. We also note that the central core-helix relationship could be used in the design of an intracellular transport motor. PMID:6788775

  5. Polyreactive Antibodies: Function and Quantification.

    PubMed

    Gunti, Sreenivasulu; Notkins, Abner Louis

    2015-07-15

    Polyreactive antibodies, a major component of the natural antibody repertoire, bind with low affinity to a variety of structurally unrelated antigens. Many of these antibodies are germline or near germline in sequence. Little is known, however, about the function of these antibodies. In the present mini-review we show: (1) that the broad antibacterial activity of the natural antibody repertoire is largely due to polyreactive antibodies, which in the presence of complement lyse bacteria and enhance phagocytosis; (2) that polyreactive antibodies bind to UV- or human immunodeficiency virus-induced apoptotic cells and with complement enhance the phagocytosis of these cells by macrophages; and (3) that dinitrophenol can be used as a surrogate for quantitating the level of polyreactive antibodies in serum. We conclude that polyreactive antibodies protect the host against both foreign invaders and its own damaged/apoptotic cells.

  6. Compositions, antibodies, asthma diagnosis methods, and methods for preparing antibodies

    DOEpatents

    Jin, Hongjun; Zangar, Richard C.

    2017-01-17

    Methods for preparing an antibody are provided with the method including incorporating 3-bromo-4-hydroxy-benzoic acid into a protein to form an antigen, immunizing a mammalian host with the antigen, and recovering an antibody having an affinity for the antigen from the host. Antibodies having a binding affinity for a monohalotyrosine are provided as well as composition comprising an antibody bound with monohalotyrosine. Compositions comprising a protein having a 3-bromo-4-hydroxy-benzoic acid moiety are also provided. Methods for evaluating the severity of asthma are provide with the methods including analyzing sputum of a patient using an antibody having a binding affinity for monohalotyrosine, and measuring the amount of antibody bound to protein. Methods for determining eosinophil activity in bodily fluid are also provided with the methods including exposing bodily fluid to an antibody having a binding affinity for monohalotyrosine, and measuring the amount of bound antibody to determine the eosinophil activity.

  7. Selection of recombinant antibodies from antibody gene libraries.

    PubMed

    Hust, Michael; Frenzel, André; Schirrmann, Thomas; Dübel, Stefan

    2014-01-01

    Antibodies are indispensable detection reagents for research and diagnostics and represent the biggest class of biological therapeutics on the market. In vitro antibody selection systems offer many advantages over animal-based technologies because the whole selection process is independent of the in vivo immune response. In the last two decades antibody phage display has evolved to the most robust and widely used method and has already yielded thousands of antibodies. The selection of binders by phage display is also referred to as "panning" and based on the specific molecular interaction of antibody phage with an immobilized antigen thus allowing the enrichment and isolation of antigen-specific monoclonal binders from very large antibody gene libraries. Here, we give detailed protocols for the selection of recombinant antibody fragments from antibody gene libraries in microtiter plates.

  8. Human germline antibody gene segments encode polyspecific antibodies.

    PubMed

    Willis, Jordan R; Briney, Bryan S; DeLuca, Samuel L; Crowe, James E; Meiler, Jens

    2013-04-01

    Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three VH gene segments. Panels of somatically mutated antibodies encoded by a common VH gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the VH gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding.

  9. Human Germline Antibody Gene Segments Encode Polyspecific Antibodies

    PubMed Central

    Willis, Jordan R.; Briney, Bryan S.; DeLuca, Samuel L.; Crowe, James E.; Meiler, Jens

    2013-01-01

    Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three VH gene segments. Panels of somatically mutated antibodies encoded by a common VH gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the VH gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding. PMID:23637590

  10. Reshaping Antibody Diversity

    PubMed Central

    Wang, Feng; Ekiert, Damian C.; Ahmad, Insha; Yu, Wenli; Zhang, Yong; Bazirgan, Omar; Torkamani, Ali; Raudsepp, Terje; Mwangi, Waithaka; Criscitiello, Michael F.; Wilson, Ian A.; Schultz, Peter G.; Smider, Vaughn V.

    2014-01-01

    Summary Unlike humans or mice, some species have limited genome encoded combinatorial diversity potential, yet mount a robust antibody response. Cows are unusual in having exceptionally long CDR H3 loops and few V-regions, but the mechanism for creating diversity is not understood. Deep sequencing revealed that ultralong CDR H3s contain a remarkable complexity of cysteines, suggesting that disulfide-bonded mini-domains may arise during repertoire development. Indeed, crystal structures of two cow antibodies reveal that these CDR H3s form a very unusual architecture composed of a β-strand “stalk” that supports a structurally diverse, disulfide-bonded, “knob” domain. Sequence analysis suggests that diversity arises from somatic hypermutation of an ultralong DH with a severe codon bias towards mutation to cysteine. These unusual antibodies can be elicited to recognize defined antigens through the knob domain. Thus, the bovine immune system produces an antibody repertoire composed of CDR H3s of unprecedented length that fold into a diversity of mini-domains generated through combinations of somatically generated disulfides. PMID:23746848

  11. Monoclonal Antibodies against Pectin

    PubMed Central

    Liners, Françoise; Letesson, Jean-Jacques; Didembourg, Christian; Van Cutsem, Pierre

    1989-01-01

    Monoclonal antibodies have been produced that recognize a conformation of homopolygalacturonic acid (pectic acid) induced by an optimum concentration of calcium and sodium of about 1 and 150 millinormal, respectively. The epitope recognized is probably part of the dimers of pectin chains associated according to the `egg box' model. Images Figure 2 PMID:16667195

  12. Monoclonal antibodies against bacteria.

    PubMed

    Macario, A J; Conway de Macario, E

    1988-01-01

    Highlights are presented of most recent work in which monoclonal antibodies have been instrumental in the study of bacteria and their products. Topics summarized pertain to human and veterinary medicines, dentistry, phytopathology, ichthyology, and bacterial ecophysiology, differentiation, evolution and methanogenic biotechnology.

  13. Recombinant monoclonal antibody technology.

    PubMed

    Siegel, D L

    2002-01-01

    With the development of murine hybridoma technology over a quarter century ago, the ability to produce large quantities of well-characterized monoclonal antibody preparations revolutionized diagnostic and therapeutic medicine. For many applications in transfusion medicine, however, the production of serological reagents in mice has certain biological limitations relating to the difficulty in obtaining murine monoclonal antibodies specific for many human blood group antigens. Furthermore, for therapeutic purposes, the efficacy of murine-derived immunoglobulin preparations is limited by the induction of anti-mouse immune responses. Technical difficulties inherent in human hybridoma formation have led to novel molecular approaches that facilitate the isolation and production of human antibodies without the need for B-cell transformation, tissue culture, or even immunized individuals. These technologies, referred to as 'repertoire cloning' or 'Fab/phage display', involve the rapid cloning of immunoglobulin gene segments to create immune libraries from which antibodies with desired specificities can be selected. The use of such recombinant methods in transfusion medicine is anticipated to play an important role in the development and production of renewable supplies of low-cost reagents for diagnostic and therapeutic applications.

  14. Therapeutic antibody engineering

    PubMed Central

    Parren, Paul W.H.I.; Lugovskoy, Alexey A.

    2013-01-01

    It is an important event in any knowledge area when an authority in the field decides that it is time to share all accumulated knowledge and learnings by writing a text book. This does not occur often in the biopharmaceutical industry, likely due to both the highly dynamic environment with tight timelines and policies and procedures at many pharmaceutical companies that hamper knowledge sharing. To take on a task like this successfully, a strong drive combined with a desire and talent to teach, but also an accommodating and stimulating environment is required. Luckily for those interested in therapeutic monoclonal antibodies, Dr. William R. Strohl decided about two years ago that the time was right to write a book about the past, present and future of these fascinating molecules. Dr. Strohl’s great expertise and passion for biotechnology is evident from his life story and his strong academic and industry track record. Dr. Strohl pioneered natural product biotechnology, first in academia as a full professor of microbiology and biochemistry at Ohio State University in Columbus, Ohio and later in industry while at Merck. Despite his notable advances in recombinant natural products, industry interest in this area waned and in 2001 Dr. Strohl sought new opportunities by entering the field of antibody therapeutics. He initiated antibody discovery through phage display at Merck, and then moved to Centocor Research and Development Inc. (now Janssen Biotech, Inc.) in 2008 to head Biologics Research, where he now directs the discovery of innovative therapeutic antibody candidates.

  15. Antibodies to cardiac receptors.

    PubMed

    Boivin-Jahns, V; Schlipp, A; Hartmann, S; Panjwani, P; Klingel, K; Lohse, M J; Ertl, G; Jahns, R

    2012-12-01

    Inflammation of cardiac tissue is generally associated with an activation of the host's immune system. On the one hand, this activation is mandatory to protect the heart by fighting the invading microbial agents or toxins and by engaging myocardial reparation and healing processes. On the other hand, uncontrolled activation of the immune defense has the risk of an arousal of auto- or cross-reactive immune cells, which in some cases bring more harm than good. Dependent on the individual genetic predisposition, such heart-directed autoimmune reactions most likely occur as a result of myocyte apoptosis or necrosis and subsequent liberation of self-antigens previously hidden to the immune system. During the past two decades, evidence for a pathogenic relevance of autoimmunity in human heart disease has substantially increased. Conformational cardiac (auto)antibodies affecting cardiac function and, in particular, (auto)antibodies that target G protein-coupled cardiac membrane receptors are thought to play a key role in the development of heart failure. Clinical pilot studies even suggest that such antibodies negatively affect survival in heart failure patients. However, the true prevalence and clinical impact of many cardiac (auto)antibodies in human heart diseases are still unclear, as are the events triggering their formation, their titer course, and their patterns of clearance and/or persistence. The present article summarizes current knowledge in the field of cardiac receptor (auto)antibodies including recent efforts to address some of the aforementioned gaps of knowledge, thereby attempting to pave the way for novel, more specific therapeutic approaches.

  16. What Is Antiphospholipid Antibody Syndrome?

    MedlinePlus

    ... AN-te-fos-fo-LIP-id) antibody syndrome (APS) is an autoimmune disorder. Autoimmune disorders occur if ... usually help defend the body against infections. In APS, however, the body makes antibodies that mistakenly attack ...

  17. Anti-smooth muscle antibody

    MedlinePlus

    ... medlineplus.gov/ency/article/003531.htm Anti-smooth muscle antibody To use the sharing features on this page, please enable JavaScript. Anti-smooth muscle antibody is a blood test that detects the ...

  18. The Art of Making Antibodies.

    ERIC Educational Resources Information Center

    Headon, Denis R.

    1986-01-01

    Provides background information for teachers on the nature and production of antibodies. Points out that the production of monoclonal antibodies blends the malignant with the beneficial to create a medical tool of exciting potential. (JN)

  19. The Art of Making Antibodies.

    ERIC Educational Resources Information Center

    Headon, Denis R.

    1986-01-01

    Provides background information for teachers on the nature and production of antibodies. Points out that the production of monoclonal antibodies blends the malignant with the beneficial to create a medical tool of exciting potential. (JN)

  20. Humanized Antibodies for Antiviral Therapy

    NASA Astrophysics Data System (ADS)

    Co, Man Sung; Deschamps, Marguerite; Whitley, Richard J.; Queen, Cary

    1991-04-01

    Antibody therapy holds great promise for the treatment of cancer, autoimmune disorders, and viral infections. Murine monoclonal antibodies are relatively easy to produce but are severely restricted for therapeutic use by their immunogenicity in humans. Production of human monoclonal antibodies has been problematic. Humanized antibodies can be generated by introducing the six hypervariable regions from the heavy and light chains of a murine antibody into a human framework sequence and combining it with human constant regions. We humanized, with the aid of computer modeling, two murine monoclonal antibodies against herpes simplex virus gB and gD glycoproteins. The binding, virus neutralization, and cell protection results all indicate that both humanized antibodies have retained the binding activities and the biological properties of the murine monoclonal antibodies.

  1. A new approach to an AIDS vaccine: creating antibodies to HIV vif will enable apobec3G to turn HIV-infection into a benign problem.

    PubMed

    Jeffrey Fessel, W

    2005-01-01

    For a decade, attempts to produce a vaccine that prevents HIV infection have been fruitless, and fresh approaches are required. Apobec3G is a natural defensin and a cytidine deaminase. Apobec3G induces a high rate of dC to dU mutation in the first minus strand of cDNA, causing degradation throughout the HIV genome that renders the virus effete. The viral infectivity factor (vif) of HIV is essential for efficient replication of that virus. Vif binds to apobec3G and induces its polyubiquitination, which enables HIV to evade apobec3G. This suggests that a vif-based vaccine which induced anti-vif antibodies, would prevent the neutralizing action of vif upon apobec3G. Then, with HIV-vif ineffective, apobec3G could act without hindrance to create a less aggressive, non-lethal HIV infection. Mutated vif impedes HIV infection. Slow progressors with vif 132S had 4-fold lower viral loads than those with vif 132R; and introducing vif 132S into HIV-1 caused a 5-fold decrease in viral replication. And in the absence of vif, HIV virions accumulate multiple defects in structural, enzymatic, and regulatory viral proteins. The success of a vif-based vaccine depends upon (1) a vif-antibody response, and (2) vif antibodies entering the cells that harbor HIV. First, antibodies to vif have been seen in frequencies ranging between 25% and 100% in patients infected with HIV-1. Second, transport of anti-vif antibodies into cells might occur via several mechanisms. Likeliest is that in viremic persons, antibodies would attach to cell-free virions which would piggyback the antibodies into CD4+ cells. Alternatively, a fusion protein between vif and a cell-surface receptor, e.g., CD4 or CCR5, might be used as vaccine antigen. Also, anti-vif antibodies might internalize after ligation of HIV virions budding on the cell surface, in the same way as monoclonal antibodies against porcine pseudorabies virus induced viral glycoproteins on the cell surface to internalize. Finally, monoclonal

  2. Antibody-gold cluster conjugates

    DOEpatents

    Hainfeld, J.F.

    1988-06-28

    Antibody- or antibody fragment-gold cluster conjugates are shown wherein the conjugate size can be about 5.0 nm. Methods and reagents are disclosed in which antibodies or Fab' fragments thereof are covalently bound to a stable cluster of gold atoms. 2 figs.

  3. Antibody response to dengue virus.

    PubMed

    Cedillo-Barrón, Leticia; García-Cordero, Julio; Bustos-Arriaga, José; León-Juárez, Moisés; Gutiérrez-Castañeda, Benito

    2014-09-01

    In this review, we discuss the current knowledge of the role of the antibody response against dengue virus and highlight novel insights into targets recognized by the human antibody response. We also discuss how the balance of pathological and protective antibody responses in the host critically influences clinical aspects of the disease. Copyright © 2014 Institut Pasteur. All rights reserved.

  4. Selection of recombinant antibodies from antibody gene libraries.

    PubMed

    Hust, Michael; Dübel, Stefan; Schirrmann, Thomas

    2007-01-01

    After the sequencing of the human genome is completed, the research focus shifts toward the analysis of gene products. The human genome encodes more than 30,000 genes. Owing to alternative mRNA splicing and posttranslational modifications, for example, glycosylation, phoshorylation, and so on, the number of different proteins of human proteome is supposed to easily exceed 90,000. Antibodies are key detection reagents for the "postgenomic" analysis of these proteins. Any systematic investigation of the human proteome requires high throughput methods for antibody generation. In vitro selection systems utilizing recombinant antibody repertoires offer this capability and capacity. The most commonly used contemporary in vitro selection system is antibody phage display, which has already yielded thousands of useful antibodies for therapy, research, and diagnostics. Herein, methods are described for the selection of recombinant antibody fragments from naive antibody gene libraries.

  5. Antibody mimetics: promising complementary agents to animal-sourced antibodies.

    PubMed

    Baloch, Abdul Rasheed; Baloch, Abdul Wahid; Sutton, Brian J; Zhang, Xiaoying

    2016-01-01

    Despite their wide use as therapeutic, diagnostic and detection agents, the limitations of polyclonal and monoclonal antibodies have inspired scientists to design the next generation biomedical agents, so-called antibody mimetics that offer many advantages over conventional antibodies. Antibody mimetics can be constructed by protein-directed evolution or fusion of complementarity-determining regions through intervening framework regions. Substantial progress in exploiting human, butterfly (Pieris brassicae) and bacterial systems to design and select mimetics using display technologies has been made in the past 10 years, and one of these mimetics [Kalbitor® (Dyax)] has made its way to market. Many challenges lie ahead to develop mimetics for various biomedical applications, especially those for which conventional antibodies are ineffective, and this review describes the current characteristics, construction and applications of antibody mimetics compared to animal-sourced antibodies. The possible limitations of mimetics and future perspectives are also discussed.

  6. Antibody Engineering and Therapeutics Conference

    PubMed Central

    Almagro, Juan Carlos; Gilliland, Gary L; Scott, Jamie; Larrick, James W; Plückthun, Andreas; Veldman, Trudi; Adams, Gregory P; Parren, Paul WHI; Chester, Kerry A; Bradbury, Andrew; Reichert, Janice M; Huston, James S

    2013-01-01

    The Antibody Engineering and Therapeutics conference, which serves as the annual meeting of The Antibody Society, will be held in Huntington Beach, CA from Sunday December 8 through Thursday December 12, 2013. The scientific program will cover the full spectrum of challenges in antibody research and development, and provide updates on recent progress in areas from basic science through approval of antibody therapeutics. Keynote presentations will be given by Leroy Hood (Institute of System Biology), who will discuss a systems approach for studying disease that is enabled by emerging technology; Douglas Lauffenburger (Massachusetts Institute of Technology), who will discuss systems analysis of cell communication network dynamics for therapeutic biologics design; David Baker (University of Washington), who will describe computer-based design of smart protein therapeutics; and William Schief (The Scripps Research Institute), who will discuss epitope-focused immunogen design.   In this preview of the conference, the workshop and session chairs share their thoughts on what conference participants may learn in sessions on: (1) three-dimensional structure antibody modeling; (2) identifying clonal lineages from next-generation data sets of expressed VH gene sequences; (3) antibodies in cardiometabolic medicine; (4) the effects of antibody gene variation and usage on the antibody response; (5) directed evolution; (6) antibody pharmacokinetics, distribution and off-target toxicity; (7) use of knowledge-based design to guide development of complementarity-determining regions and epitopes to engineer or elicit the desired antibody; (8) optimizing antibody formats for immunotherapy; (9) antibodies in a complex environment; (10) polyclonal, oligoclonal and bispecific antibodies; (11) antibodies to watch in 2014; and (12) polyreactive antibodies and polyspecificity.

  7. Antibody-mediated radiotherapy

    SciTech Connect

    Bloomer, W.D.; Lipsztein, R.; Dalton, J.F.

    1985-05-01

    Antibodies that react with antigens on the surface of tumor cells but not normal cells have great potential for cancer detection and therapy. If radiolabeled without loss of immunologic specificity, such antibodies may be able to deliver cytoxic amounts of radiation. Target- cell specificity and a high extraction coefficient are necessary with any radionuclide in order to minimize normal tissue irradiation. Tumor- cell-retention time and the rate of catabolized radionuclide will also influence ultimate applicability. Among the unanswered questions for choosing a radionuclide is the choice of particle emitter. Although classic beta emitters have been used in a number of clinical situations, they have not had a major impact on disease outcome except in diseases of the thyroid. Unfortunately, Auger emitters such as iodine 125 are cytotoxic only when localized within close proximity to the genome. On the other hand, alpha emitters such as astatine 211 eliminate the need for subcellular sequestration but not cell-specific localization. 34 references.

  8. Antibody Therapy for Histoplasmosis

    PubMed Central

    Nosanchuk, Joshua D.; Zancopé-Oliveira, Rosely M.; Hamilton, Andrew J.; Guimarães, Allan J.

    2012-01-01

    The endemic human pathogenic fungus Histoplasma capsulatum is a major fungal pathogen with a broad variety of clinical presentations, ranging from mild, focal pulmonary disease to life-threatening systemic infections. Although azoles, such as itraconazole and voriconazole, and amphotericin B have significant activity against H. capsulatum, about 1 in 10 patients hospitalized due to histoplasmosis die. Hence, new approaches for managing disease are being sought. Over the past 10 years, studies have demonstrated that monoclonal antibodies (mAbs) can modify the pathogenesis of histoplasmosis. Disease has been shown to be impacted by mAbs targeting either fungal cell surface proteins or host co-stimulatory molecules. This review will detail our current knowledge regarding the impact of antibody therapy on histoplasmosis. PMID:22347215

  9. Construction of human naive antibody gene libraries.

    PubMed

    Hust, Michael; Frenzel, André; Meyer, Torsten; Schirrmann, Thomas; Dübel, Stefan

    2012-01-01

    Human antibodies are valuable tools for proteome research and diagnostics. Furthermore, antibodies are a rapidly growing class of therapeutic agents, mainly for inflammation and cancer therapy. The first therapeutic antibodies are of murine origin and were chimerized or humanized. The later-developed antibodies are fully human antibodies. Here, two technologies are competing the hybridoma technology using transgenic mice with human antibody gene loci and antibody phage display. The starting point for the selection of human antibodies against any target is the construction of an antibody phage display gene library.In this review we describe the construction of human naive and immune antibody gene libraries for antibody phage display.

  10. Antibodies Targeting EMT

    DTIC Science & Technology

    2015-10-01

    epithelial colonies. The single clones were grown under 4- OHT induction as well as blasticidine selection as mentioned above. The genomic DNA was...of the genes back into the lentiviral vector and sequencing single clones. Optimization of the isolation of the genomic DNA , optimization of primers...01/2015 – 06/30/2017 “Antibody Fingerprinting of Triple Negative Breast Cancer by High Throughput FACS” The goal of this research is to use a novel

  11. Antibodies Targeting EMT

    DTIC Science & Technology

    2015-10-01

    colonies. The single clones were grown under 4- OHT induction as well as blasticidine selection as mentioned above. The genomic DNA was extracted from...genes back into the lentiviral vector and sequencing single clones. Optimization of the isolation of the genomic DNA , optimization of primers, and...01/2015 – 06/30/2017 “Antibody Fingerprinting of Triple Negative Breast Cancer by High Throughput FACS” The goal of this research is to use a

  12. [Antibody therapy for Alzheimer's disease].

    PubMed

    Tabira, Takeshi; Matsumoto, Shin-Ei; Jin, Haifeng

    2011-11-01

    In order to avoid Abeta-induced autoimmune encephalitis, several monoclonal and polyclonal antibodies are in clinical trials. These are bapineuzumab, solanezumab, ponezumab, gantenerumab, BAN2401, gammaguard and octagam. Since each antibody has a different antigen epitope of Abeta, anti-amyloid activities are different. It is unknown which antibody is effective for Alzheimer disease, and we must wait for the result of clinical trials. Some patients who developed tissue amyloid plaque immuno-reactive (TAPIR) antibody showed slower decline after AN-1792 vaccination. We developed TAPIR-like monoclonal antibody, which was found to react with Abeta oligomers preferentially.

  13. Monoclonal antibodies to gonadotropin subunits

    SciTech Connect

    Ehrlich, P.H.; Moyle, W.R.; Canfield, R.E.

    1985-01-01

    The production of monoclonal antibodies to peptide hormones, with their unifocal binding sites, can provide tools for understanding hormone structure and function. The paper focuses on techniques that are important for the study of monoclonal antibodies to chorionic gonadotropin (hCG), including hybridoma production, methods of screening for desired clones, properties of the monoclonal antibodies, effect of antibodies on hormone-receptor interaction, inhibition of binding of radiolabeled hCG, inhibition of hCG induced steroidogenesis, determination of relative orientation of epitopes, and synergistic actions of monoclonal antibodies to hCG.

  14. Antibodies for immunoassays.

    PubMed

    Newman, D J

    2000-01-01

    What is an immunoassay without an antibody? Clearly the name provides the answer to this question; without antibodies there would be no immunoassays. An immunoassay is an analytical technique, quantitative or qualitative, that relies absolutely on the specificity and affinity of the interaction between epitope and paratope for generation of a detectable response. The actual detection of this binding interaction can be via one of literally hundreds of different signal transduction mechanisms, e.g., fluorimetry, chemiluminescence, agglutination (turbidimetry or nephelometry) enzyme reactions, and so forth (1 -4), but these are simply transducing systems for the primary binding interaction. Antibodies thus provide us with an exquisitely sensitive and specific analytical technology for detecting and quantifying epitopic structures. These structures include amino-acid derivatives, e.g., thyroid hormones, peptides, e.g., vasopressin, proteins, e.g., cytokines, as well as carbohydrate structures, e.g., CA-125. Immunoassay technology has developed to such an extent that it is probably the most versatile analytical tool available able to identify and quantify epitopic structures across the milli- to zeptomolar concentration ranges (2).

  15. Antineutrophil cytoplasmic antibodies.

    PubMed

    Bosch, Xavier; Guilabert, Antonio; Font, Josep

    2006-07-29

    Much like other autoantibodies (eg, anti-double stranded DNA in systemic lupus erythematosus or antiglomerular basement membrane antibodies in Goodpasture's syndrome), antineutrophil cytoplasmic antibodies (ANCA) have provided doctors with a useful serological test to assist in diagnosis of small-vessel vasculitides, including Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, and their localised forms (eg, pauci-immune necrotising and crescentic glomerulonephritis). 85-95% of patients with Wegener's granulomatosis, microscopic polyangiitis, and pauci-immune necrotising and crescentic glomerulonephritis have serum ANCA. ANCA directed to either proteinase 3 or myeloperoxidase are clinically relevant, yet the relevance of other ANCA remains unknown. Besides their diagnostic potential, ANCA might be valuable in disease monitoring. In addition, data seem to confirm the long-disputed pathogenic role of these antibodies. Present treatments for ANCA-associated vasculitis are not free from side-effects and as many as 50% of patients relapse within 5 years. Accurate understanding of the key pathogenic points of ANCA-associated vasculitis can undoubtedly provide a more rational therapeutic approach.

  16. Antibody therapy for Ebola

    PubMed Central

    Qiu, Xiangguo; Kobinger, Gary P

    2014-01-01

    Ebola viruses can cause severe hemorrhagic fever in humans and nonhuman primates with fatality rates up to 90%, and are identified as biosafety level 4 pathogens and CDC Category A Agents of Bioterrorism. To date, there are no approved therapies and vaccines available to treat these infections. Antibody therapy was estimated to be an effective and powerful treatment strategy against infectious pathogens in the late 19th, early 20th centuries but has fallen short to meet expectations to widely combat infectious diseases. Passive immunization for Ebola virus was successful in 2012, after over 15 years of failed attempts leading to skepticism that the approach would ever be of potential benefit. Currently, monoclonal antibody (mAbs)-based therapies are the most efficient at reversing the progression of a lethal Ebola virus infection in nonhuman primates, which recapitulate the human disease with the highest similarity. Novel combinations of mAbs can even fully cure lethally infected animals after clinical symptoms and circulating virus have been detected, days into the infection. These new developments have reopened the door for using antibody-based therapies for filovirus infections. Furthermore, they are reigniting hope that these strategies will contribute to better control the spread of other infectious agents and provide new tools against infectious diseases. PMID:24503566

  17. A monoclonal antibody against leptin.

    PubMed

    Mahmoudian, Jafar; Jeddi-Tehrani, Mahmood; Bayat, Ali Ahmad; Mahmoudi, Ahmad Reza; Vojgani, Yasaman; Tavangar, Banafsheh; Hadavi, Reza; Zarei, Saeed

    2012-10-01

    Leptin is an important protein that regulates energy storage and homeostasis in humans and animals. Leptin deficiency results in various abnormalities such as diabetes, obesity, and infertility. Producing a high affinity monoclonal antibody against human leptin provides an important tool to monitor and trace leptin function in different biological fluids. In this study, recombinant human leptin was conjugated to KLH and injected into mice. After immunization, mouse myeloma SP2/0 cells were fused with murine splenocytes followed by selection of antibody-producing hybridoma cells. After screening of different hybridoma colonies by ELISA, a high affinity antibody was selected and purified by affinity chromatography. The affinity constant of the antibody was measured by ELISA. Western blot, immunocytochemistry, and flow cytometry experiments were used to characterize the antibody. The anti-leptin antibody had a high affinity (around 1.13 × 10(-9) M) for its antigen. The saturation of the antibody with leptin (20 moles leptin per 1 mole antibody) in Western blot analysis proved that the antibody had specific binding to its antigen. Immunocytochemistry and flow cytometry on JEG-3 (human placental choriocarcinoma cell) cells revealed that the anti-leptin antibody recognized intracellular leptin. In conclusion, we report here the production and characterization of a murine anti-leptin antibody with high affinity for human leptin.

  18. Microbials for the production of monoclonal antibodies and antibody fragments

    PubMed Central

    Spadiut, Oliver; Capone, Simona; Krainer, Florian; Glieder, Anton; Herwig, Christoph

    2014-01-01

    Monoclonal antibodies (mAbs) and antibody fragments represent the most important biopharmaceutical products today. Because full length antibodies are glycosylated, mammalian cells, which allow human-like N-glycosylation, are currently used for their production. However, mammalian cells have several drawbacks when it comes to bioprocessing and scale-up, resulting in long processing times and elevated costs. By contrast, antibody fragments, that are not glycosylated but still exhibit antigen binding properties, can be produced in microbial organisms, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression systems, strain engineering, and production processes for the three main microbials used in antibody and antibody fragment production, namely Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. PMID:24183828

  19. Antibody Therapy for Pediatric Leukemia

    PubMed Central

    Vedi, Aditi; Ziegler, David S.

    2014-01-01

    Despite increasing cure rates for pediatric leukemia, relapsed disease still carries a poor prognosis with significant morbidity and mortality. Novel targeted therapies are currently being investigated in an attempt to reduce adverse events and improve survival outcomes. Antibody therapies represent a form of targeted therapy that offers a new treatment paradigm. Monoclonal antibodies are active in pediatric acute lymphoblastic leukemia (ALL) and are currently in Phase III trials. Antibody-drug conjugates (ADCs) are the next generation of antibodies where a highly potent cytotoxic agent is bound to an antibody by a linker, resulting in selective targeting of leukemia cells. ADCs are currently being tested in clinical trials for pediatric acute myeloid leukemia and ALL. Bispecific T cell engager (BiTE) antibodies are a construct whereby each antibody contains two binding sites, with one designed to engage the patient’s own immune system and the other to target malignant cells. BiTE antibodies show great promise as a novel and effective therapy for childhood leukemia. This review will outline recent developments in targeted agents for pediatric leukemia including monoclonal antibodies, ADCs, and BiTE antibodies. PMID:24795859

  20. [Antiglycolipid antibody in inflammatory neuropathy].

    PubMed

    Kusunoki, S

    1995-12-01

    Antiglycolipid antibody is frequently detected in the acute phase sera from patients with acute inflammatory demyelinating polyneuropathy (Guillain-Barré syndrome, GBS). The titer is highest in the serum sample taken first after the neurological onset, and decreases with clinical improvement. Antiglycolipid antibody may play a role in the pathogenetic mechanism of GBS. GM1 and GD1b are the antigens most commonly recognized. Monoclonal anti-GD1b antibody specifically bound to the paranodal myelin of the peripheral nervous system. Serum anti-GD1b antibody may cause demyelinative neuropathy by binding to the paranodal myelin of the peripheral nervous system. Anti-GQ1b IgG antibody is specifically raised in almost all the sera from Fisher syndrome and GBS with ophthalmoplegia. Anti-GQ1b monoclonal antibody immunostained specifically the paranodal myelin of the extramedullary portion of oculomotor, trochlear and abducens nerves, but no such staining was observed in the other peripheral nerves. Anti-GQ1b antibody may cause conduction block in the cranial nerves innervating the muscles for extraocular movement by binding to the paranodal myelin of those nerves. Anti-GalNAc-GD1a antibody is detected in the patients with GBS with very low or inexcitable compound muscle action potentials. The sera from patients with GBS subsequent to mycoplasma infection had antigalactocerebroside antibody. Further study on antiglycolipid antibody is needed for understanding the pathogenetic mechanism of GBS.

  1. From rabbit antibody repertoires to rabbit monoclonal antibodies

    PubMed Central

    Weber, Justus; Peng, Haiyong; Rader, Christoph

    2017-01-01

    In this review, we explain why and how rabbit monoclonal antibodies have become outstanding reagents for laboratory research and increasingly for diagnostic and therapeutic applications. Starting with the unique ontogeny of rabbit B cells that affords highly distinctive antibody repertoires rich in in vivo pruned binders of high diversity, affinity and specificity, we describe the generation of rabbit monoclonal antibodies by hybridoma technology, phage display and alternative methods, along with an account of successful humanization strategies. PMID:28336958

  2. How antibodies use complement to regulate antibody responses.

    PubMed

    Sörman, Anna; Zhang, Lu; Ding, Zhoujie; Heyman, Birgitta

    2014-10-01

    Antibodies, forming immune complexes with their specific antigen, can cause complete suppression or several 100-fold enhancement of the antibody response. Immune complexes containing IgG and IgM may activate complement and in such situations also complement components will be part of the immune complex. Here, we review experimental data on how antibodies via the complement system upregulate specific antibody responses. Current data suggest that murine IgG1, IgG2a, and IgG2b upregulate antibody responses primarily via Fc-receptors and not via complement. In contrast, IgM and IgG3 act via complement and require the presence of complement receptors 1 and 2 (CR1/2) expressed on both B cells and follicular dendritic cells. Complement plays a crucial role for antibody responses not only to antigen complexed to antibodies, but also to antigen administered alone. Lack of C1q, but not of Factor B or MBL, severely impairs antibody responses suggesting involvement of the classical pathway. In spite of this, normal antibody responses are found in mice lacking several activators of the classical pathway (complement activating natural IgM, serum amyloid P component (SAP), specific intracellular adhesion molecule-grabbing non-integrin R1 (SIGN-R1) or C-reactive protein. Possible explanations to these observations will be discussed.

  3. Human antibody technology and the development of antibodies against cytomegalovirus.

    PubMed

    Ohlin, Mats; Söderberg-Nauclér, Cecilia

    2015-10-01

    Cytomegalovirus (CMV) is a virus that causes chronic infections in a large set of the population. It may cause severe disease in immunocompromised individuals, is linked to immunosenescence and implied to play an important role in the pathogenesis of cardiovascular diseases and cancer. Modulation of the immune system's abilities to manage the virus represent a highly viable therapeutic option and passive immunotherapy with polyclonal antibody preparations is already in clinical use. Defined monoclonal antibodies offer many advantages over polyclonal antibodies purified from serum. Human CMV-specific monoclonal antibodies have consequently been thoroughly investigated with respect to their potential in the treatment of diseases caused by CMV. Recent advances in human antibody technology have substantially expanded the breadth of antibodies for such applications. This review summarizes the fundamental basis for treating CMV disease by use of antibodies, the basic technologies to be used to develop such antibodies, and relevant human antibody specificities available to target this virus. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Understanding antinuclear antibodies.

    PubMed

    Pertusi, R M; Rubin, B R

    1991-06-01

    The American College of Rheumatology (formerly the American Rheumatism Association) diagnostic criteria for connective tissue disorders frequently include positive antinuclear antibody (ANA) assays. Proper interpretation of these tests requires an understanding of the principles governing ANA assays. Assay results are reported in two ways: as titers and as descriptions of fluorescent patterns. A titer is a quantitative measure of ANAs in serum. Different patterns of immunofluorescence are associated with different subsets of collagen vascular disease. Positive results can occur in the absence of connective tissue disease. Accurate diagnosis of connective tissue disorders requires judicious use of ANA assays as well as skillful interpretation of the results.

  5. Antibody Glossary —

    Cancer.gov

    The components of the immune system have diverse roles in the initial development of cancers, progression of early-stage malignancies to invasive tumors, establishment of metastatic lesions, tumor dormancy, and response or resistance to therapy. Characterizing the components of the immune system and their functional status in tissues and in tumors requires the use of highly specific reagents. Researchers employ antibodies in a variety of in vitro and in vivo applications to delineate, enrich, or deplete specific immune subsets in order to understand their role(s) in tumorigenesis. This is a glossary of validated reagents and protocols that are useful for functional phenotyping of the immune system in murine cancer models.

  6. Antibody response in Heterodontus.

    PubMed

    Litman, G W; Erickson, B W; Lederman, L; Mäkelä, O

    1982-05-28

    Appropriately selected phylogenetic models are capable of providing insight into genetic mechanisms which may have become obscured during the passage of evolutionary time. In higher vertebrates a complex multigenic family encodes immunoglobulin-variable regions. The mechanisms involved in the expansion of the gene family and the stable maintenance of large numbers of individual genes presently are not understood. By defining the nature of antibody diversity in lower vertebrate species, it may be possible to approach such issues at a more fundamental level. Analyses of the immunoglobulins in Heterodontus francisci (horned shark), a representative phylogenetically primitive elasmobranch, indicate that this species may represent a useful developmental model.

  7. Guinea-pig reaginic antibody

    PubMed Central

    Margni, R. A.; Hajos, Silvia E.

    1973-01-01

    The physicochemical and biological properties of purified guinea-pig reaginic antibody were studied. It is a labile protein different to γ1. Its antibody activity is completely destroyed by heating at 56° for 6 hours and by treatment with mercaptoethanol. The capacity to give PCA is decreased by repeated freezing and thawing. It is a bivalent antibody, haemagglutinating, does not fix complement and is capable of sensitizing guinea-pig skin for PCA reaction after a latent period of a week but not after 3 hours. Reaginic antibody appears on day 7–8 after the first inoculation and the higher levels (PCA reaction) are obtained at the eleventh to thirteenth days. After the fifteenth to seventeenth days the PCA is negative. The reaginic antibody does not pass the placenta. Higher levels of reaginic antibody were obtained when the guinea-pigs were inoculated with the antigen in saline with simultaneous inoculation, intraperitoneally, of killed Bordetella pertussis, phase I. PMID:4354828

  8. Monoclonal antibodies for treating cancer

    SciTech Connect

    Dillman, R.O. )

    1989-10-01

    The purpose of this study is to assess the current status of in-vivo use of monoclonal antibodies for treating cancer. Publications appearing between 1980 and 1988 were identified by computer searches using MEDLINE and CANCERLIT, by reviewing the table of contents of recently published journals, and by searching bibliographies of identified books and articles. More than 700 articles, including peer-reviewed articles and book chapters, were identified and selected for analysis. The literature was reviewed and 235 articles were selected as relevant and representative of the current issues and future applications for in-vivo monoclonal antibodies for cancer therapy and of the toxicity and efficacy which has been associated with clinical trials. Approaches include using antibody alone (interacting with complement or effector cells or binding directly with certain cell receptors) and immunoconjugates (antibody coupled to radioisotopes, drugs, toxins, or other biologicals). Most experience has been with murine antibodies. Trials of antibody alone and radiolabeled antibodies have confirmed the feasibility of this approach and the in-vivo trafficking of antibodies to tumor cells. However, tumor cell heterogeneity, lack of cytotoxicity, and the development of human antimouse antibodies have limited clinical efficacy. Although the immunoconjugates are very promising, heterogeneity and the antimouse immune response have hampered this approach as has the additional challenge of chemically or genetically coupling antibody to cytotoxic agents. As a therapeutic modality, monoclonal antibodies are still promising but their general use will be delayed for several years. New approaches using human antibodies and reducing the human antiglobulin response should facilitate treatment. 235 references.

  9. Monoclonal antibodies to Actinobacillus actinomycetemcomitans.

    PubMed Central

    Place, D A; Scidmore, N C; McArthur, W P

    1988-01-01

    Murine hybridoma cell lines were developed which synthesized monoclonal antibodies against Actinobacillus actinomycetemcomitans-associated antigens. Monoclonal antibodies specific for an antigen(s) common to all A. actinomycetemcomitans isolates tested but not detected on other gram-negative oral plaque microorganisms or other Actinobacillus species were identified. Monoclonal antibodies specific for each serotype group of A. actinomycetemcomitans which did not bind to other Actinobacillus species or oral plaque microorganisms were also identified. PMID:3356470

  10. [Antineutrophil cytoplasmic antibodies].

    PubMed

    Sebastiani, G D

    2009-01-01

    Antineutrophil cytoplasmic antibodies (ANCA) are predominantly IgG autoantibodies directed against constituents of primary granules of neutrophils and monocytes lysosomes. Although several antigenic targets have been identified, those ANCA directed to proteinase 3 or myeloperoxidase are clinically relevant, whereas the importance of other ANCA remains unknown. Both are strongly associated with small vessel vasculitides, the ANCA-associated vasculitides, which include Wegener's granulomatosis, microscopic polyangiitis, and Churg-Strauss syndrome, and the localised forms of these diseases (eg, pauci-immune necrotising and crescentic glomerulonephritis). ANCA is a useful serological test to assist in diagnosis of small-vessel vasculitides. 85-95% of patients with Wegener's granulomatosis, microscopic polyangiitis, and pauci-immune necrotising and crescentic glomerulonephritis have serum ANCA. ANCA directed to either proteinase 3 or myeloperoxidase are clinically relevant, yet the relevance of other ANCA remains unknown. Besides their diagnostic potential, ANCA might be valuable in disease monitoring. In addition, data seem to confirm the long-disputed pathogenic role of these antibodies. There is increasing evidence that myeloperoxidase-ANCA are directly involved in the pathogenesis of necrotizing vasculitis. This is less clear for proteinase 3-ANCA, markers for Wegener's granulomatosis. With respect to proteinase 3-ANCA, complementary proteinase 3, a peptide translated from the antisense DNA strand of proteinase 3 and homologous to several microbial peptides, may be involved in induction of proteinase 3-antineutrophil cytoplasmic autoantibodies.

  11. Serosurvey for polio antibodies.

    PubMed

    Biberi-Moroeanu, S; Munţiu, A; Stoiculescu, S

    1988-01-01

    From 1982 to 1988 a serological survey was performed on 573 subjects aged 3-80 years in order to evaluate the polio immunity level of the population. The presence of neutralizing antibodies was tested using the types 1, 2 and 3 Sabin vaccine strains as well as a wild strain of poliovirus type 1 isolated in France in 1978. According to their birth date, the subjects were assigned to 4 groups. The differences between the groups consisted in the different vaccination history of the subjects as well as in their different opportunities of having been in contact with the past epidemics of poliomyelitis. The results obtained indicate a satisfactory polio immunity level in all the 4 groups: seropositives, 96.7%-98.9% for type 2, 91.8%-98.2% for type 1 (Sabin vaccine strain), 89.3%-96.6% for type 3 and 84.2%-96.4% for type 1 wild strain. The highest immunity levels were found in group D (children with recorded history of complete polio vaccination) and in group A (unvaccinated people but contemporary with the past polio epidemics). A special comment is made with respect to 14 subjects showing satisfactory antibody titres for all the three types of Sabin-vaccine strains but who have proved to be seronegative (less than 4) for the wild type 1 poliovirus strain.

  12. Monoclonal antibodies and neuroblastoma

    SciTech Connect

    Miraldi, F. )

    1989-10-01

    Several antineuroblastoma monoclonal antibodies (MoAbs) have been described and two have been used in radioimmunoimaging and radioimmunotherapy in patients. MoAb 3F8 is a murine IgG3 antibody specific for the ganglioside GD2. Radioiodine-labeled 3F8 has been shown to specifically target human neuroblastoma in patients, and radioimmunoimaging with this agent has provided consistently high uptakes with tumor-to-background ratios of greater than or equal to 10:1. Radioimmunotherapy has been attempted with both MoAb 3F8 and MoAb UJ13A, and although encouraging results have been obtained, dosimetry data and tissue dose response information for these agents is lacking, which impedes the development of such therapy. 124I, a positron emitter, can be used with 3F8 in positron emission tomography (PET) scanning to provide dosimetry information for radioimmunotherapy. The tumor radiation dose response from radiolabeled MoAb also can be followed with PET images with fluorodeoxyglucose (FDG) scanning of neuroblastoma tumors. Results to date indicate that radioimmunoimaging has clinical use in the diagnosis of neuroblastoma and the potential for radioimmunotherapy for this cancer remains high.48 references.

  13. Human anti-mouse antibodies.

    PubMed

    Klee, G G

    2000-06-01

    Human anti-mouse antibodies (HAMA) are human immunoglobulins with specificity for mouse immunoglobulins. This topic currently is of interest because of the increased use of monoclonal mouse antibodies as diagnostic reagents both for in vitro laboratory measurements and for in vivo imaging studies. Monoclonal mouse antibodies also are being used therapeutically. This short article reviews the production of HAMA in patients receiving monoclonal antibodies and illustrates the potential ways that HAMA can interfere with immunoassay measurements. Methods for measuring and neutralizing HAMA also are discussed.

  14. Antibody therapies in CNS diseases.

    PubMed

    Freskgård, Per-Ola; Urich, Eduard

    2017-07-01

    Therapeutic antibodies have essentially been banned from the central nervous system, and are so far limited to use mainly in multiple sclerosis. This is primarily due to the fact that antibody penetration across the blood-brain barrier is very limited, with about only 0.1% of circulating antibodies estimated to reach the brain at steady-state concentration. Nonetheless, advances are being made with conventional antibodies, showing that minimal exposure can act centrally to mediate therapeutic effects. Immunotherapy in Alzheimer's disease is a noteworthy example where antibodies against amyloid-β are able to reduce brain plaque pathology in preclinical models and humans. However, the advances in using antibodies directed at brain targets have also demonstrated impediments of low brain exposure in achieving clinical benefits, spurring increased attention in technologies designed to improve brain exposure of antibodies. Here we review antibodies in clinical trials for central nervous system disorders. Moreover, we describe some of the efforts to improve the therapeutic efficacy of antibodies by enhancing delivery across the blood-brain barrier. This article is part of the Special Issue entitled "Beyond small molecules for neurological disorders". Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Antibodies as stratagems against cancer.

    PubMed

    Papageorgiou, Louis; Cuong, Nguyen Tien; Vlachakis, Dimitrios

    2016-06-21

    Antibodies have been in the frontline of anticancer research during the last few decades, since a number of different ways have been discovered to utilize them as parts or main components of anticancer drugs. Antibodies are used as the only component of some anticancer drugs, but they can also be conjugated with a variety of substances. Antibody engineering methods such as humanization, chimerization and Fc engineering are applied in order to modify their properties according to the requirements of anticancer drug application. Given the continuous advances in biology and informatics, the role of antibodies in anticancer treatment is expected to be prominent.

  16. Creating Ordered Antibody Arrays with Antibody-Polymer Conjugates

    NASA Astrophysics Data System (ADS)

    Dong, Xuehui; Obermeyer, Allie; Olsen, Bradley

    Antibodies are a category of functional proteins that play crucial roles in the immune system and have been widely applied in the area of cancer therapeutics, targeting delivery, signal detection, and sensors. Due to the extremely large size and lack of specific functional groups on the surface, it is challenging to functionalize antibodies and manipulate the ordered packing of antibodies in an array with high density and proper orientation, which is critical to achieve outstanding performance in materials. In this work, we demonstrate an efficient and facile approach for preparing antibody-polymer conjugates with two-step sequential ``click'' reaction to form antibody-polymer block copolymers. Highly ordered nanostructures are fabricated based on the principles of block copolymer self-assembly. The nanostructures are studied with both small angle X-ray scattering (SAXS) and transmission electron microscopy (TEM). Lamellae with alternating antibody domain and polymer domain are observed with an overall domain size of ~50 nm. The nanostructure not only increases the packing density and promotes proper orientation of the antibody, but also provides possible channel to facilitate substrate transportation and improves the stability of the antibody.

  17. Endotoxin Tolerance Impairs IL-1 Receptor-Associated Kinase (IRAK) 4 and TGF-β-activated Kinase 1 Activation, K63-linked Polyubiquitination and Assembly of IRAK1, TNF Receptor-associated Factor 6, and IκB Kinase γ and Increases A20 Expression*

    PubMed Central

    Xiong, Yanbao; Qiu, Fu; Piao, Wenji; Song, Chang; Wahl, Larry M.; Medvedev, Andrei E.

    2011-01-01

    Endotoxin tolerance reprograms Toll-like receptor 4 responses by impairing LPS-elicited production of pro-inflammatory cytokines without inhibiting expression of anti-inflammatory or anti-microbial mediators. In septic patients, Toll-like receptor tolerance is thought to underlie decreased pro-inflammatory cytokine expression in response to LPS and increased incidence of microbial infections. The impact of endotoxin tolerance on recruitment, post-translational modifications and signalosome assembly of IL-1 receptor-associated kinase (IRAK) 4, IRAK1, TNF receptor-associated factor (TRAF) 6, TGF-β-activated kinase (TAK) 1, and IκB kinase (IKK) γ is largely unknown. We report that endotoxin tolerization of THP1 cells and human monocytes impairs LPS-mediated receptor recruitment and activation of IRAK4, ablates K63-linked polyubiquitination of IRAK1 and TRAF6, compromises assembly of IRAK1-TRAF6 and IRAK1-IKKγ platforms, and inhibits TAK1 activation. Deficiencies in these signaling events in LPS-tolerant cells coincided with increased expression of A20, an essential deubiquitination enzyme, and sustained A20-IRAK1 associations. Overexpression of A20 inhibited LPS-induced activation of NF-κB and ablated NF-κB reporter activation driven by ectopic expression of MyD88, IRAK1, IRAK2, TRAF6, and TAK1/TAB1, while not affecting the responses induced by IKKβ and p65. A20 shRNA knockdown abolished LPS tolerization of THP1 cells, mechanistically linking A20 and endotoxin tolerance. Thus, deficient LPS-induced activation of IRAK4 and TAK1, K63-linked polyubiquitination of IRAK1 and TRAF6, and disrupted IRAK1-TRAF6 and IRAK1-IKKγ assembly associated with increased A20 expression and A20-IRAK1 interactions are new determinants of endotoxin tolerance. PMID:21220427

  18. Endotoxin tolerance impairs IL-1 receptor-associated kinase (IRAK) 4 and TGF-beta-activated kinase 1 activation, K63-linked polyubiquitination and assembly of IRAK1, TNF receptor-associated factor 6, and IkappaB kinase gamma and increases A20 expression.

    PubMed

    Xiong, Yanbao; Qiu, Fu; Piao, Wenji; Song, Chang; Wahl, Larry M; Medvedev, Andrei E

    2011-03-11

    Endotoxin tolerance reprograms Toll-like receptor 4 responses by impairing LPS-elicited production of pro-inflammatory cytokines without inhibiting expression of anti-inflammatory or anti-microbial mediators. In septic patients, Toll-like receptor tolerance is thought to underlie decreased pro-inflammatory cytokine expression in response to LPS and increased incidence of microbial infections. The impact of endotoxin tolerance on recruitment, post-translational modifications and signalosome assembly of IL-1 receptor-associated kinase (IRAK) 4, IRAK1, TNF receptor-associated factor (TRAF) 6, TGF-β-activated kinase (TAK) 1, and IκB kinase (IKK) γ is largely unknown. We report that endotoxin tolerization of THP1 cells and human monocytes impairs LPS-mediated receptor recruitment and activation of IRAK4, ablates K63-linked polyubiquitination of IRAK1 and TRAF6, compromises assembly of IRAK1-TRAF6 and IRAK1-IKKγ platforms, and inhibits TAK1 activation. Deficiencies in these signaling events in LPS-tolerant cells coincided with increased expression of A20, an essential deubiquitination enzyme, and sustained A20-IRAK1 associations. Overexpression of A20 inhibited LPS-induced activation of NF-κB and ablated NF-κB reporter activation driven by ectopic expression of MyD88, IRAK1, IRAK2, TRAF6, and TAK1/TAB1, while not affecting the responses induced by IKKβ and p65. A20 shRNA knockdown abolished LPS tolerization of THP1 cells, mechanistically linking A20 and endotoxin tolerance. Thus, deficient LPS-induced activation of IRAK4 and TAK1, K63-linked polyubiquitination of IRAK1 and TRAF6, and disrupted IRAK1-TRAF6 and IRAK1-IKKγ assembly associated with increased A20 expression and A20-IRAK1 interactions are new determinants of endotoxin tolerance.

  19. Antibodies: Protective, destructive and regulatory role

    SciTech Connect

    Milgrom, F.; Abeyounis, C.J.; Albini, B.

    1985-01-01

    This book contains papers under 10 subject headings. The headings are: Production and Function of Antibodies, Protective Role of Antibodies, Antibodies to Foreign and Neoplastic Cells, Autoantibodies, Regulatory Mechanisms, Allergy, Immune Complexes, Antibodies in Pregnancy and Aging, Administration of Antibodies for Prevention and Therapy, and Abstracts of Poster Presentations.

  20. Micromechanical antibody sensor

    DOEpatents

    Thundat, Thomas G.; Jacobson, K. Bruce; Doktycz, Mitchel J.; Kennel, Stephen J.; Warmack, Robert J.

    2001-01-01

    A sensor apparatus is provided using a microcantilevered spring element having a coating of a detector molecule such as an antibody or antigen. A sample containing a target molecule or substrate is provided to the coating. The spring element bends in response to the stress induced by the binding which occurs between the detector and target molecules. Deflections of the cantilever are detected by a variety of detection techniques. The microcantilever may be approximately 1 to 200 .mu.m long, approximately 1 to 50 .mu.m wide, and approximately 0.3 to 3.0 .mu.m thick. A sensitivity for detection of deflections is in the range of 0.01 nanometers.

  1. New engineered antibodies against prions

    PubMed Central

    Škrlj, Nives; Dolinar, Marko

    2014-01-01

    A number of recently developed and approved therapeutic agents based on highly specific and potent antibodies have shown the potential of antibody therapy. As the next step, antibody-based therapeutics will be bioengineered in a way that they not only bind pathogenic targets but also address other issues, including drug targeting and delivery. For antibodies that are expected to act within brain tissue, like those that are directed against the pathogenic prion protein isoform, one of the major obstacles is the blood-brain barrier which prevents efficient transfer of the antibody, even of the engineered single-chain variants. We recently demonstrated that a specific prion-specific antibody construct which was injected into the murine tail vein can be efficiently transported into brain tissue. The novelty of the work was in that the cell penetrating peptide was used as a linker connecting both specificity-determining domains of the antibody peptide, thus eliminating the need for the standard flexible linker, composed of an arrangement of three consecutive (Gly4Ser) repeats. This paves the road toward improved bioengineered antibody variants that target brain antigens. PMID:23941991

  2. [The significance of antiphospholipid antibodies].

    PubMed

    Fojtík, Z

    2004-04-01

    Antiphospholipid antibodies (APLA) present very heterogeneous groups of antibodies which can significantly influence processes on different levels of coagulation cascade depending on effects of phospholipid surfaces on blood coagulation. This usually leads to a particular level of thrombophylia. Clinical syndrome accompanying positive APLA, such as antiphospholipid syndrome, was defined by clinical and laboratory symptoms. This clinical syndrome can be a primary syndrome, if other disorders with ability to induce generation of antibodies can be excluded, or a secondary syndrome. The most often in cases of systemic tissue disease. APLA can be divided according to the presence of lupus anticoagulant and anticardiolipin antibodies. According to a definition lupus anticoagulants are antibodies able to inhibit and prolong in vitro one or more blood clotting processes dependent on phospholipid surfaces. Anticardiolipin antibodies are antibodies measured by ELISA method with cardiolipin used as an antibody. Findings show that some APLA are directed against proteins bound to phospholipid surfaces. Main cofactor proteins include beta 2-GPI and prothrombin. Because of their heterogeneous specificity, APLA are directed against negative phospholipids or proteins bound to phospholipid surfaces and have important pathophysiology role in development of antiphospholipid syndrome.

  3. Heterogeneous Antibody Responses in Tuberculosis

    PubMed Central

    Lyashchenko, Konstantin; Colangeli, Roberto; Houde, Michel; Al Jahdali, Hamdan; Menzies, Dick; Gennaro, Maria Laura

    1998-01-01

    Antibody responses during tuberculosis were analyzed by an enzyme-linked immunosorbent assay with a panel of 10 protein antigens of Mycobacterium tuberculosis. It was shown that serum immunoglobulin G antibodies were produced against a variety of M. tuberculosis antigens and that the vast majority of sera from tuberculosis patients contained antibodies against one or more M. tuberculosis antigens. The number and the species of serologically reactive antigens varied greatly from individual to individual. In a given serum, the level of specific antibodies also varied with the antigen irrespective of the total number of antigens recognized by that particular serum. These findings indicate that person-to-person heterogeneity of antigen recognition, rather than recognition of particular antigens, is a key attribute of the antibody response in tuberculosis. PMID:9673283

  4. Metrics for antibody therapeutics development.

    PubMed

    Reichert, Janice M

    2010-01-01

    A wide variety of full-size monoclonal antibodies (mAbs) and therapeutics derived from alternative antibody formats can be produced through genetic and biological engineering techniques. These molecules are now filling the preclinical and clinical pipelines of every major pharmaceutical company and many biotechnology firms. Metrics for the development of antibody therapeutics, including averages for the number of candidates entering clinical study and development phase lengths for mAbs approved in the United States, were derived from analysis of a dataset of over 600 therapeutic mAbs that entered clinical study sponsored, at least in part, by commercial firms. The results presented provide an overview of the field and context for the evaluation of on-going and prospective mAb development programs. The expansion of therapeutic antibody use through supplemental marketing approvals and the increase in the study of therapeutics derived from alternative antibody formats are discussed.

  5. Antinuclear antibodies in localized scleroderma.

    PubMed

    Takehara, K; Moroi, Y; Nakabayashi, Y; Ishibashi, Y

    1983-05-01

    When HeLa cells were used as the substrate for detection by the indirect immunofluorescence method, antinuclear antibodies were demonstrated in 16 of 22 (72.7%) sera from patients with localized scleroderma. When mouse kidney sections were used, the positive rate for antinuclear antibodies was 50% (11 of 22). In the 3 subgroups of localized scleroderma, frequencies of antinuclear antibodies on HeLa cells were as follows: morphea, 50% (2 of 4), generalized morphea, 100% (6 of 6), linear scleroderma, 67% (8 of 12). Antibodies to centromere, Scl-70, nuclear RNP, Sm, and SS-B antigens were not detected in any patients with localized scleroderma. The high frequency of antinuclear antibodies in localized scleroderma sera suggests that localized scleroderma is a disease which, though different from diffuse scleroderma, also involves an immunologic abnormality.

  6. Endogenous Antibodies for Tumor Detection

    PubMed Central

    Rich, Barrie S.; Honeyman, Joshua N.; Darcy, David G.; Smith, Peter T.; Williams, Andrew R.; Lim, Irene Isabel P.; Johnson, Linda K.; Gönen, Mithat; Simon, Joel S.; LaQuaglia, Michael P.; Simon, Sanford M.

    2014-01-01

    The study of cancer immunology has provided diagnostic and therapeutic instruments through serum autoantibody biomarkers and exogenous monoclonal antibodies. While some endogenous antibodies are found within or surrounding transformed tissue, the extent to which this exists has not been entirely characterized. We find that in transgenic and xenograft mouse models of cancer, endogenous gamma immunoglobulin (IgG) is present at higher concentration in malignantly transformed organs compared to non-transformed organs in the same mouse or organs of cognate wild-type mice. The enrichment of endogenous antibodies within the malignant tissue provides a potential means of identifying and tracking malignant cells in vivo as they mutate and diversify. Exploiting these antibodies for diagnostic and therapeutic purposes is possible through the use of agents that bind endogenous antibodies. PMID:24875800

  7. Radiolabeled antibodies in cancer. Oncology Overview

    SciTech Connect

    Not Available

    1984-11-01

    Oncology Overviews are a service of the International Cancer Research Data Bank (ICRDB) Program of the National Cancer Institute, intended to facilitate and promote the exchange of information between cancer scientists by keeping them aware of literature related to their research being published by other laboratories through the world. Each Oncology Overview represents a survey of the literature associated with a selected area of cancer research. It contains abstracts of articles which have been selected and organized by researchers associated with the field. Contents: Radiolabeled antibodies--labeling and imaging techniques; Radiolabeled antibodies--carcinoembryonic antigen; Radiolabeled antibodies--alpha-fetoprotein; Radiolabeled antibodies--human chorionic gonadotropin; Radiolabeled antibodies--ferritin; Radiolabeled antibodies--imaging of colorectal tumors; Radiolabeled antibodies--imaging of malignant melanoma; Radiolabeled antibodies--imaging of urogenital tumors; Radiolabeled antibodies--imaging of thyroid tumors; Radiolabeled antibodies--other clinical studies; Radiolabeled antibodies--selected preclinical studies; Radiolabeled antibodies--reviews.

  8. Antibodies - Office of Cancer Clinical Proteomics Research

    Cancer.gov

    NCI announces the release of monoclonal antipeptide antibodies from rabbit for distribution on the antibody portal. There are 60 recently added monoclonal antibodies, with 56 generated from mouse and 4 generated from rabbit.

  9. Antibodies to Phospholipids and Liposomes: Binding of Antibodies to Cells

    DTIC Science & Technology

    1987-01-01

    LIPOSOMES: BINDING OF ANTIBODIES TO CELLS 12. PERSONAL AUTHOR(S) W.E. FOGLER , G. M. SWARTZ, AND C.R. ALVING 13a TYPE OF REPORT 13b. TIME COVERED 14. DATE...Elsevier BBA 73693 Antibodies to phospholipids and liposomes: binding of antibodies to cells William E. Fogler *, Glenn M. Swartz, Jr. and Carl R. Alving...Immunol. 21. Research Associateship from the U.S. National 12863-86812Hall. T. and Esser, K. (1984) 3. Immunol. 132. 2059-2063 Research Council. 13 Fogler

  10. The making of bispecific antibodies

    PubMed Central

    2017-01-01

    ABSTRACT During the past two decades we have seen a phenomenal evolution of bispecific antibodies for therapeutic applications. The ‘zoo’ of bispecific antibodies is populated by many different species, comprising around 100 different formats, including small molecules composed solely of the antigen-binding sites of two antibodies, molecules with an IgG structure, and large complex molecules composed of different antigen-binding moieties often combined with dimerization modules. The application of sophisticated molecular design and genetic engineering has solved many of the technical problems associated with the formation of bispecific antibodies such as stability, solubility and other parameters that confer drug properties. These parameters may be summarized under the term ‘developability’. In addition, different ‘target product profiles’, i.e., desired features of the bispecific antibody to be generated, mandates the need for access to a diverse panel of formats. These may vary in size, arrangement, valencies, flexibility and geometry of their binding modules, as well as in their distribution and pharmacokinetic properties. There is not ‘one best format’ for generating bispecific antibodies, and no single format is suitable for all, or even most of, the desired applications. Instead, the bispecific formats collectively serve as a valuable source of diversity that can be applied to the development of therapeutics for various indications. Here, a comprehensive overview of the different bispecific antibody formats is provided. PMID:28071970

  11. Encephalitis and GABAB receptor antibodies

    PubMed Central

    Höftberger, Romana; Titulaer, Maarten J.; Sabater, Lidia; Dome, Balazs; Rózsás, Anita; Hegedus, Balazs; Hoda, Mir Alireza; Laszlo, Viktoria; Ankersmit, Hendrik Jan; Harms, Lutz; Boyero, Sabas; de Felipe, Alicia; Saiz, Albert; Dalmau, Josep

    2013-01-01

    Objective: To report the clinical features of 20 newly diagnosed patients with GABAB receptor (GABABR) antibodies and determine the frequency of associated tumors and concurrent neuronal autoantibodies. Methods: Clinical data were retrospectively obtained and evaluated. Serum and CSF samples were examined for additional antibodies using methods previously reported. Results: Seventeen patients presented with seizures, memory loss, and confusion, compatible with limbic encephalitis (LE), one patient presented with ataxia, one patient presented with status epilepticus, and one patient presented with opsoclonus-myoclonus syndrome (OMS). Nineteen (95%) patients eventually developed LE during the course of the disease. Small-cell lung cancer (SCLC) was identified in 10 (50%) patients, all with LE. Treatment and outcome was available from 19 patients: 15 showed complete (n = 7) or partial (n = 8) neurologic improvement after steroids, IV immunoglobulins, or plasma exchange and oncologic treatment when indicated; 1 patient died of tumor progression shortly after the first cycle of immunotherapy, and 3 were not treated. Five patients with SCLC had additional onconeuronal antibodies (Ri, amphiphysin, or SOX1), and 2 without tumor had GAD65 and NMDAR antibodies, respectively. GABABR antibodies were not detected in serum of 116 patients with SCLC without neurologic symptoms. Conclusion: Our study confirms GABABR as an autoantigen of paraneoplastic and nonparaneoplastic LE and expands the phenotype of GABABR antibodies to ataxia, OMS, and status epilepticus. The long-term prognosis is dictated by the presence of a tumor. Recognition of syndromes associated with GABABR antibodies is important because they usually respond to treatment. PMID:24068784

  12. Production systems for recombinant antibodies.

    PubMed

    Schirrmann, Thomas; Al-Halabi, Laila; Dübel, Stefan; Hust, Michael

    2008-05-01

    Recombinant antibodies are the fastest growing class of therapeutic proteins. Furthermore, antibodies are key detection reagents in research and diagnostics. The increasing demand for antibodies with regards to amount and quality resulted in the development of a variety of recombinant production systems employing gram-negative and gram-positive bacteria, yeast and filamentous fungi, insect cell lines as well as mammalian cell lines. More recently, antibodies were also successfully produced in transgenic plants and animals. Currently, the production of recombinant antibodies for therapy is performed in mammalian cell lines to reduce the risk of immunogenicity caused by non-human post-translational modifications, in particular glycosylation. However, novel strategies already allow human-like glycosylation patterns in yeast, insect cell lines and transgenic plants. Furthermore, therapeutic strategies not requiring glycosylation of the Fc portion have been conceived, most prominently using bispecific antibodies or scFv fusion proteins, which can be produced in bacteria. Here, we review all current antibody production systems considering their advantages and limitations with respect to intended applications.

  13. Antibody replacement therapy in primary antibody deficiencies and iatrogenic hypogammaglobulinemia.

    PubMed

    Hoffman, Thijs W; van Kessel, Diana A; van Velzen-Blad, Heleen; Grutters, Jan C; Rijkers, Ger T

    2015-01-01

    Antibody replacement therapy has been used in the treatment of primary antibody deficiencies (PADs) for several decades, and an evidence-based guideline for its treatment is currently available. By contrast, the use of antibody replacement therapy in iatrogenic hypogammaglobulinemia (IHG), a condition that is associated with immunosuppressive medication, has hardly any evidence base and no guidelines. As IHG can be equally as severe as PAD and is much more prevalent, evidence-based guidelines are urgently needed. This review will focus on the differences and similarities between PAD and IHG and the use of antibody replacement therapy in both conditions. Suggestions for the development of evidence-based guidelines and future research are given.

  14. STUDIES ON ANTIBODY PRODUCTION

    PubMed Central

    Sainte-Marie, Guy; Coons, Albert H.

    1964-01-01

    Cells from lymph nodes of rabbits injected repeatedly with bovine serum albumin were transferred subcutaneously to previously irradiated rabbits, and the recipients were immediately injected with bovine serum albumin. A good antibody response resulted. In a series of such animals killed on successive days, skin samples at sites of cell deposition were removed and examined by immunofluorescence and by light microscopy. In these tissues abundant plasmocytes were found to have multiplied and differentiated in a regular progression from immature, to medium, to mature plasmocytes. During the 6 days of the experiment the small plasmocytes accumulated until they reached 85 per cent of the total plasmocytic population. The mitotic index of the large and medium plasmocytes averaged 11 per cent, implying a generation time of 6.3 hours on the basis of a 1 hour mitotic time. This rate of growth is sufficiently rapid to account for all the plasmocytes on the 6th day as deriving from less than 1 per cent of the population initially transferred. This rate and the orderly progression in the evolution of the plasmocytic population, make it highly improbable that plasmocytes arise from transformation of lymphocytes, but rather indicate that they spring from specific precursors already present among the transferred cells. PMID:14157028

  15. Pneumococcal Antibody Titers

    PubMed Central

    Abghari, Pamella F.; Poowuttikul, Pavadee; Secord, Elizabeth

    2017-01-01

    Purpose: Immunoglobulin replacement is the mainstay treatment in patients with humoral immunodeficiencies, yet a handful of patients continue to develop sinopulmonary infections while on therapy. The objective of our study was to compare immunoglobulin G (IgG) pneumococcal antibody levels in patients with humoral immune deficiencies who have been on intravenous immunoglobulin (IVIG) replacement for at least 1 year to those on subcutaneous immunoglobulin (SCIG) therapy for at least 1 year. Methods: A retrospective chart review was completed on 28 patients. These patients’ ages ranged between 1 and 61 years. Pneumococcal serotype titers obtained at least 1 year after initiating therapy were compared between patients on IVIG (19 patients) and SCIG (9 patients). Results: A comparison between the groups demonstrated that SCIG achieved a higher percentage of serotype titers protective for noninvasive disease (≥1.3) and 100% protection for invasive disease (≥0.2). Our data also demonstrated a similar lack of protection (less than 50% ≥1.3) in 9N, 12F, and 23F on IVIG and 4, 9N, 12F, and 23F on SCIG. Conclusions: Our data demonstrated that serotypes 1, 3, 4, 9N, 12F, and 23F exhibited the lowest random IgG means while on IVIG, which was comparable to other published studies that looked at the mean IgG levels. In addition, our retrospective chart review demonstrated a greater number of therapeutic pneumococcal titers with SCIG in comparison to IVIG. PMID:28321436

  16. [Soluble elastin and antibody formation].

    PubMed

    Stoklasová, A; Randová, Z; Wimmerová, J; Ledvina, M

    1991-01-01

    In the present study, we have obtained antibodies in rabbits to peptides prepared by digestion of elastin with either oxalic acid or phosphoric acid. By Elisa assay, we have shown the lowest production of antibodies against elastin-derived peptides prepared by digestion of N-elastin with phosphoric acid. We have found a strong cross-reactivity between antibodies obtained to peptides prepared by different digestion of insoluble elastin from bovine ligamentum nuchae or aorta and these antigens. A less pronounced cross-reactivity was observed in case of elastin-derived peptides from porcine aorta.

  17. Reducing heterophilic antibody interference in immunoassays using single chain antibodies

    SciTech Connect

    Baird, Cheryl L.; Tan, Ruimin; Fischer, Christopher J.; Victry, Kristin D.; Zangar, Richard C.; Rodland, Karin D.

    2011-12-15

    Sandwich ELISA microarrays have the potential to simultaneously quantify the levels of multiple diagnostic targets in a biological sample. However, as seen with traditional ELISA diagnostics, heterophilic antibodies (HA) in patient sera have the potential to cause interference in these assays. We demonstrate here that reducing the diagnostic capture antibody to its minimal functional unit, the variable heavy and light domains artificially connected with a short polypeptide linker (scFv), is an effective strategy for reducing the HA assay interference.

  18. Construction of human antibody gene libraries and selection of antibodies by phage display.

    PubMed

    Schirrmann, Thomas; Hust, Michael

    2010-01-01

    Recombinant antibodies as therapeutics offer new opportunities for the treatment of many tumor diseases. To date, 18 antibody-based drugs are approved for cancer treatment and hundreds of anti-tumor antibodies are under development. The first clinically approved antibodies were of murine origin or human-mouse chimeric. However, since murine antibody domains are immunogenic in human patients and could result in human anti-mouse antibody (HAMA) responses, currently mainly humanized and fully human antibodies are developed for therapeutic applications.Here, in vitro antibody selection technologies directly allow the selection of human antibodies and the corresponding genes from human antibody gene libraries. Antibody phage display is the most common way to generate human antibodies and has already yielded thousands of recombinant antibodies for research, diagnostics and therapy. Here, we describe methods for the construction of human scFv gene libraries and the antibody selection.

  19. Surface chemistries for antibody microarrays

    SciTech Connect

    Seurynck-Servoss, Shannon L.; Baird, Cheryl L.; Rodland, Karin D.; Zangar, Richard C.

    2007-05-01

    Enzyme-linked immunosorbent assay (ELISA) microarrays promise to be a powerful tool for the detection of disease biomarkers. The original technology for printing ELISA microarray chips and capturing antibodies on slides was derived from the DNA microarray field. However, due to the need to maintain antibody structure and function when immobilized, surface chemistries used for DNA microarrays are not always appropriate for ELISA microarrays. In order to identify better surface chemistries for antibody capture, a number of commercial companies and academic research groups have developed new slide types that could improve antibody function in microarray applications. In this review we compare and contrast the commercially available slide chemistries, as well as highlight some promising recent advances in the field.

  20. Antineutrophil cytoplasmic antibodies (ANCA).

    PubMed

    Radice, A; Sinico, R A

    2005-02-01

    Antineutrophil cytoplasmic antibodies (ANCA) are a sensitive and specific marker for ANCA-associated systemic vasculitis. Using indirect immunofluorescence on ethanol-fixed neutrophils, two major fluoroscopic patterns can be recognised: a diffuse cytoplasmic staining (C-ANCA), and a perinuclear/nuclear staining (P-ANCA). In patients with vasculitis, more of 90% of C-ANCA are directed against proteinase 3 (PR3-ANCA) whereas approximately 80-90% of P-ANCA recognise myelperoxidase (MPO-ANCA). Although C-ANCA (PR3-ANCA) is preferentially associated with Wegener's granulomatosis (WG), and P-ANCA (MPO-ANCA) with microscopic polyangiitis (MPA), idiopathic necrotising crescentic glomerulonephritis (iNCGN) and Churg-Strauss syndrome (CSS), there is not absolute specificity. Between 10-20% of patients with classical WG show P-ANCA (MPO-ANCA), and even a larger percentage of patients with MPA or CSS have C-ANCA (PR3-ANCA). Furthermore, it should be stressed that approximately 10-20% of patients with WG or MPA (and 40-50% of cases of CSS) have negative assay for ANCA. The best diagnostic performance is obtained when indirect immunofluorescence is combined with PR3 and MPO-specific ELISAs. ANCA with different and unknown antigen specificity are found in a variety of conditions other than AASV, including inflammatory bowel diseases, other autoimmune diseases, and infections where their clinical significance is unclear. ANCA levels are useful to monitor disease activity but should not be used by themselves to guide treatment. A significant increase in ANCA titres, or the reappearance of ANCA, should alert the clinicians and lead to a stricter patient control.

  1. Fragmentation of monoclonal antibodies

    PubMed Central

    Vlasak, Josef

    2011-01-01

    Fragmentation is a degradation pathway ubiquitously observed in proteins despite the remarkable stability of peptide bond; proteins differ only by how much and where cleavage occurs. The goal of this review is to summarize reports regarding the non-enzymatic fragmentation of the peptide backbone of monoclonal antibodies (mAbs). The sites in the polypeptide chain susceptible to fragmentation are determined by a multitude of factors. Insights are provided on the intimate chemical mechanisms that can make some bonds prone to cleavage due to the presence of specific side-chains. In addition to primary structure, the secondary, tertiary and quaternary structures have a significant impact in modulating the distribution of cleavage sites by altering local flexibility, accessibility to solvent or bringing in close proximity side chains that are remote in sequence. This review focuses on cleavage sites observed in the constant regions of mAbs, with special emphasis on hinge fragmentation. The mechanisms responsible for backbone cleavage are strongly dependent on pH and can be catalyzed by metals or radicals. The distribution of cleavage sites are different under acidic compared to basic conditions, with fragmentation rates exhibiting a minimum in the pH range 5–6; therefore, the overall fragmentation pattern observed for a mAb is a complex result of structural and solvent conditions. A critical review of the techniques used to monitor fragmentation is also presented; usually a compromise has to be made between a highly sensitive method with good fragment separation and the capability to identify the cleavage site. The effect of fragmentation on the function of a mAb must be evaluated on a case-by-case basis depending on whether cleavage sites are observed in the variable or constant regions, and on the mechanism of action of the molecule. PMID:21487244

  2. Avian Diagnostic and Therapeutic Antibodies

    SciTech Connect

    Bradley, David Sherman

    2012-12-31

    A number of infectious agents have the potential of causing significant clinical symptomology and even death, but dispite this, the number of incidence remain below the level that supports producing a vaccine. Therapeutic antibodies provide a viable treatment option for many of these diseases. We proposed that antibodies derived from West Nile Virus (WNV) immunized geese would be able to treat WNV infection in mammals and potential humans. We demonstrated that WNV specific goose antibodies are indeed successful in treating WNV infection both prophylactically and therapeutically in a golden hamster model. We demonstrated that the goose derived antibodies are non-reactogenic, i.e. do not cause an inflammatory response with multiple exposures in mammals. We also developed both a specific pathogen free facility to house the geese during the antibody production phase and a patent-pending purification process to purify the antibodies to greater than 99% purity. Therefore, the success of these study will allow a cost effective rapidly producible therapeutic toward clinical testing with the necessary infrastructure and processes developed and in place.

  3. Neutralising Antibodies against Ricin Toxin

    PubMed Central

    Prigent, Julie; Panigai, Laetitia; Lamourette, Patricia; Sauvaire, Didier; Devilliers, Karine; Plaisance, Marc; Volland, Hervé; Créminon, Christophe; Simon, Stéphanie

    2011-01-01

    The Centers for Disease Control and Prevention have listed the potential bioweapon ricin as a Category B Agent. Ricin is a so-called A/B toxin produced by plants and is one of the deadliest molecules known. It is easy to prepare and no curative treatment is available. An immunotherapeutic approach could be of interest to attenuate or neutralise the effects of the toxin. We sought to characterise neutralising monoclonal antibodies against ricin and to develop an effective therapy. For this purpose, mouse monoclonal antibodies (mAbs) were produced against the two chains of ricin toxin (RTA and RTB). Seven mAbs were selected for their capacity to neutralise the cytotoxic effects of ricin in vitro. Three of these, two anti-RTB (RB34 and RB37) and one anti-RTA (RA36), when used in combination improved neutralising capacity in vitro with an IC50 of 31 ng/ml. Passive administration of association of these three mixed mAbs (4.7 µg) protected mice from intranasal challenges with ricin (5 LD50). Among those three antibodies, anti-RTB antibodies protected mice more efficiently than the anti-RTA antibody. The combination of the three antibodies protected mice up to 7.5 hours after ricin challenge. The strong in vivo neutralising capacity of this three mAbs combination makes it potentially useful for immunotherapeutic purposes in the case of ricin poisoning or possibly for prevention. PMID:21633505

  4. Novel antibodies as anticancer agents.

    PubMed

    Zafir-Lavie, I; Michaeli, Y; Reiter, Y

    2007-05-28

    In recent years antibodies, whether generated by traditional hybridoma technology or by recombinant DNA strategies, have evolved from Paul Ehrlich's 'magic bullets' to a modern age 'guided missile'. In the recent years of immunologic research, we are witnessing development in the fields of antigen screening and protein engineering in order to create specific anticancer remedies. The developments in the field of recombinant DNA, protein engineering and cancer biology have let us gain insight into many cancer-related mechanisms. Moreover, novel techniques have facilitated tools allowing unique distinction between malignantly transformed cells, and regular ones. This understanding has paved the way for the rational design of a new age of pharmaceuticals: monoclonal antibodies and their fragments. Antibodies can select antigens on both a specific and a high-affinity account, and further implementation of these qualities is used to target cancer cells by specifically identifying exogenous antigens of cancer cell populations. The structure of the antibody provides plasticity resonating from its functional sites. This review will screen some of the many novel antibodies and antibody-based approaches that are being currently developed for clinical applications as the new generation of anticancer agents.

  5. Antibodies to watch in 2015

    PubMed Central

    Reichert, Janice M

    2015-01-01

    The commercial pipeline of recombinant antibody therapeutics is robust and dynamic. As of early December 2014, a total of 6 such products (vedolizumab, siltuximab, ramucirumab, pembrolizumab, nivolumab, blinatumomab) were granted first marketing approvals in 2014. As discussed in this perspective on antibodies in late-stage development, the outlook for additional approvals, potentially still in 2014 and certainly in 2015, is excellent as marketing applications for 6 antibody therapeutics (secukinumab, evolocumab, mepolizumab, dinutuximab, nivolumab, necitumumab) are undergoing a first regulatory review in the EU or US. Of the 39 novel mAbs currently in Phase 3 studies, a marketing application for one (alirocumab) may be submitted in late 2014, and marketing application submissions for at least 4 (reslizumab, ixekizumab, ocrelizumab, obiltoxaximab) are expected in 2015. Other ‘antibodies to watch’ are those in Phase 3 studies with estimated primary completion dates in late 2014 or 2015, which includes 13 for non-cancer indications (brodalumab, bimagrumab, bococizumab, MABp1, gevokizumab, dupilumab, sirukumab, sarilumab, tildrakizumab, guselkumab, epratuzumab, combination of actoxumab + bezlotoxumab, romosozumab) and 2 (racotumomab and clivatuzumab tetraxetan) undergoing evaluation as treatments for cancer. In addition to the novel antibody therapeutics mentioned, biosimilar infliximab and biosimilar trastuzumab are ‘antibodies to watch’ in 2015 because of their potential for entry into the US market and regulatory review, respectively. PMID:25484055

  6. Antibodies to watch in 2015.

    PubMed

    Reichert, Janice M

    2015-01-01

    The commercial pipeline of recombinant antibody therapeutics is robust and dynamic. As of early December 2014, a total of 6 such products (vedolizumab, siltuximab, ramucirumab, pembrolizumab, nivolumab, blinatumomab) were granted first marketing approvals in 2014. As discussed in this perspective on antibodies in late-stage development, the outlook for additional approvals, potentially still in 2014 and certainly in 2015, is excellent as marketing applications for 7 antibody therapeutics (secukinumab, evolocumab, mepolizumab, dinutuximab, nivolumab, blinatumomab, necitumumab) are undergoing a first regulatory review in the EU or US. Of the 39 novel mAbs currently in Phase 3 studies, a marketing application for one (alirocumab) may be submitted in late 2014, and marketing application submissions for at least 4 (reslizumab, ixekizumab, ocrelizumab, obiltoxaximab) are expected in 2015. Other 'antibodies to watch' are those in Phase 3 studies with estimated primary completion dates in late 2014 or 2015, which includes 13 for non-cancer indications (brodalumab, bimagrumab, bococizumab, MABp1, gevokizumab, dupilumab, sirukumab, sarilumab, tildrakizumab, guselkumab, epratuzumab, combination of actoxumab + bezlotoxumab, romosozumab) and 2 (racotumomab and clivatuzumab tetraxetan) undergoing evaluation as treatments for cancer. In addition to the novel antibody therapeutics mentioned, biosimilar infliximab and biosimilar trastuzumab are 'antibodies to watch' in 2015 because of their potential for entry into the US market and regulatory review, respectively.

  7. [The pharmacokinetics of monoclonal antibodies].

    PubMed

    Keizer, R J; Huitema, A D R; Damen, C W N; Schellens, J H M; Beijnen, J H

    2007-03-24

    Monoclonal antibodies (MOABs) are, due to their specificity, increasingly being deployed for therapeutic purposes. MOABs are derived from immunoglobulins and are fully or partially of murine or human origin. They are administered parenterally: mostly intravenously, but subcutaneous or intramuscular administration is also possible, in which case absorption probably occurs through the lymphatic system. The distribution of MOABs from the bloodstream into the tissues is slow and is hampered by the high molecular size of the MOABs, which is a lesser problem for fragments of antibodies (Fab fragments). MOABs are metabolised to peptides and amino acids. This process takes place in many tissues of the body, but probably predominantly in epithelial cells. As a consequence of the saturable binding of the MOAB to its target, a dose-dependent (non-linear) elimination is often observed. Immune reactions can accelerate the elimination of antibodies, partially depending on the degree ofhumanisation of the antibody. Antibodies and endogenous immunoglobulins are protected from elimination by binding to protective receptors (neonatal Fc-receptor; FcRn), which explains their long half-lives (up to 4 weeks). Metabolic pharmacokinetic interactions with other drugs have not been reported and are not expected. It is expected that in the years to come, new MOABs directed towards new targets will appear on the market, as well as existing antibodies with improved pharmacokinetic properties.

  8. Replacing reprogramming factors with antibodies selected from combinatorial antibody libraries.

    PubMed

    Blanchard, Joel W; Xie, Jia; El-Mecharrafie, Nadja; Gross, Simon; Lee, Sohyon; Lerner, Richard A; Baldwin, Kristin K

    2017-09-11

    The reprogramming of differentiated cells into induced pluripotent stem cells (iPSCs) is usually achieved by exogenous induction of transcription by factors acting in the nucleus. In contrast, during development, signaling pathways initiated at the membrane induce differentiation. The central idea of this study is to identify antibodies that can catalyze cellular de-differentiation and nuclear reprogramming by acting at the cell surface. We screen a lentiviral library encoding ∼100 million secreted and membrane-bound single-chain antibodies and identify antibodies that can replace either Sox2 and Myc (c-Myc) or Oct4 during reprogramming of mouse embryonic fibroblasts into iPSCs. We show that one Sox2-replacing antibody antagonizes the membrane-associated protein Basp1, thereby de-repressing nuclear factors WT1, Esrrb and Lin28a (Lin28) independent of Sox2. By manipulating this pathway, we identify three methods to generate iPSCs. Our results establish unbiased selection from autocrine combinatorial antibody libraries as a robust method to discover new biologics and uncover membrane-to-nucleus signaling pathways that regulate pluripotency and cell fate.

  9. Engineered antibodies take center stage.

    PubMed

    Huston, J S; George, A J

    2001-01-01

    The start of the post-genomic era provides a useful juncture for reflection on the state of antibody engineering, which will be a critical technology for relating function and pathology to genomic sequence in biology and medicine. The phenomenal progress in deciphering the human genome has given significant impetus to the application of engineered antibodies in proteomics. Thus, advances in phage display antibody libraries can now help to define novel gene function and the measurement of abnormal protein expression in pathological states. Furthermore, intrabody and antibody engineering provide vehicles for the development of molecular medicines of the future. In addition to these new directions, antibody engineering has begun to show concrete success in its long-term efforts to develop targeted immunotherapies for cancer and other diseases. The cornerstones of clinical development are the detailed academic clinical trials that continue to push the boundaries of engineered antibodies into the real world. The field displays a healthy impatience for practical results, as research accelerates with concerted efforts to transfer preclinical insights into clinical trials. Growing private and governmental expenditures will lead to the rapid expansion of life-saving immunotherapeutic agents. The present review developed from our effort to report on the 11th Annual International Conference on Antibody Engineering (3-6 December 2000). This annual meeting is a forum for discussions on the latest advances in antibody engineering groups from around the world, and now includes the broader agenda of engineering in molecular immunology. In bringing scientists together to exchange ideas at this open forum, new collaborations and the threads of new discoveries are woven. For example, Professors Gerhard Wagner (Harvard Medical School), Dennis Burton (Scripps Research Institute), and Peter Hudson (CSIRO, Melbourne, Australia) gave exciting insights on structural immunobiology that had

  10. Validating Antibodies to the Cannabinoid CB2 Receptor: Antibody Sensitivity Is Not Evidence of Antibody Specificity.

    PubMed

    Marchalant, Yannick; Brownjohn, Philip W; Bonnet, Amandine; Kleffmann, Torsten; Ashton, John C

    2014-06-01

    Antibody-based methods for the detection and quantification of membrane integral proteins, in particular, the G protein-coupled receptors (GPCRs), have been plagued with issues of primary antibody specificity. In this report, we investigate one of the most commonly utilized commercial antibodies for the cannabinoid CB2 receptor, a GPCR, using immunoblotting in combination with mass spectrometry. In this way, we were able to develop powerful negative and novel positive controls. By doing this, we are able to demonstrate that it is possible for an antibody to be sensitive for a protein of interest-in this case CB2-but still cross-react with other proteins and therefore lack specificity. Specifically, we were able to use western blotting combined with mass spectrometry to unequivocally identify CB2 protein in over-expressing cell lines. This shows that a common practice of validating antibodies with positive controls only is insufficient to ensure antibody reliability. In addition, our work is the first to develop a label-free method of protein detection using mass spectrometry that, with further refinement, could provide unequivocal identification of CB2 receptor protein in native tissues.

  11. Antibodies to watch in 2016.

    PubMed

    Reichert, Janice M

    2016-01-01

    The number of novel antibody therapeutics that received first marketing approvals in 2015 met expectations, with 6 (alirocumab (Praluent®), evolocumab (Repatha®), daratumumab (Darzalex®), dinutuximab (Unituxin®), idarucizumab (Praxbind®), mepolizumab (Nucala®)) granted first approvals as of mid-November*. Seven novel antibody therapeutics (begelomab, brodalumab, elotuzumab, ixekizumab, necitumumab, obiltoxaximab, reslizumab) are in regulatory review, and thus a similar number, if not more, are projected to gain first approvals in 2016. Commercial late-stage antibody therapeutics development exceeded expectations by increasing from 39 candidates in Phase 3 studies as of late 2014 to 53 as of late 2015. Of the 53 candidates, transitions to regulatory review by the end of 2016 are projected for 8 (atezolizumab, benralizumab, bimagrumab, durvalumab, inotuzumab ozogamicin, lebrikizumab, ocrelizumab, tremelimumab). Other "antibodies to watch" include 15 candidates (bavituximab, bococizumab, dupilumab, fasinumab, fulranumab, gevokizumab, guselkumab, ibalizumab, LY2951742, onartuzumab, REGN2222, roledumab, romosozumab, sirukumab, Xilonix) undergoing evaluation in Phase 3 studies that have estimated primary completion dates in 2016. As evidenced by the antibody therapeutics discussed in this perspective, the biopharmaceutical industry has a highly active late-stage clinical pipeline that may deliver numerous new products to the global market in the near future. *See Note added in proof for updates through December 31, 2015.

  12. Antibodies: an alternative for antibiotics?

    PubMed

    Berghman, L R; Abi-Ghanem, D; Waghela, S D; Ricke, S C

    2005-04-01

    In 1967, the success of vaccination programs, combined with the seemingly unstoppable triumph of antibiotics, prompted the US Surgeon General to declare that "it was time to close the books on infectious diseases." We now know that the prediction was overly optimistic and that the fight against infectious diseases is here to stay. During the last 20 yr, infectious diseases have indeed made a staggering comeback for a variety of reasons, including resistance against existing antibiotics. As a consequence, several alternatives to antibiotics are currently being considered or reconsidered. Passive immunization (i.e., the administration of more or less pathogen-specific antibodies to the patient) prior to or after exposure to the disease-causing agent is one of those alternative strategies that was almost entirely abandoned with the introduction of chemical antibiotics but that is now gaining interest again. This review will discuss the early successes and limitations of passive immunization, formerly referred to as "serum therapy," the current use of antibody administration for prophylaxis or treatment of infectious diseases in agriculture, and, finally, recent developments in the field of antibody engineering and "molecular farming" of antibodies in various expression systems. Especially the potential of producing therapeutic antibodies in crops that are routine dietary components of farm animals, such as corn and soy beans, seems to hold promise for future application in the fight against infectious diseases.

  13. Antibodies to watch in 2016

    PubMed Central

    Reichert, Janice M.

    2016-01-01

    ABSTRACT The number of novel antibody therapeutics that received first marketing approvals in 2015 met expectations, with 6 (alirocumab (Praluent®), evolocumab (Repatha®), daratumumab (Darzalex®), dinutuximab (Unituxin®), idarucizumab (Praxbind®), mepolizumab (Nucala®)) granted first approvals as of mid-November*. Seven novel antibody therapeutics (begelomab, brodalumab, elotuzumab, ixekizumab, necitumumab, obiltoxaximab, reslizumab) are in regulatory review, and thus a similar number, if not more, are projected to gain first approvals in 2016. Commercial late-stage antibody therapeutics development exceeded expectations by increasing from 39 candidates in Phase 3 studies as of late 2014 to 53 as of late 2015. Of the 53 candidates, transitions to regulatory review by the end of 2016 are projected for 8 (atezolizumab, benralizumab, bimagrumab, durvalumab, inotuzumab ozogamicin, lebrikizumab, ocrelizumab, tremelimumab). Other "antibodies to watch" include 15 candidates (bavituximab, bococizumab, dupilumab, fasinumab, fulranumab, gevokizumab, guselkumab, ibalizumab, LY2951742, onartuzumab, REGN2222, roledumab, romosozumab, sirukumab, Xilonix) undergoing evaluation in Phase 3 studies that have estimated primary completion dates in 2016. As evidenced by the antibody therapeutics discussed in this perspective, the biopharmaceutical industry has a highly active late-stage clinical pipeline that may deliver numerous new products to the global market in the near future. *See Note added in proof for updates through December 31, 2015. PMID:26651519

  14. Molecular-specific urokinase antibodies

    NASA Technical Reports Server (NTRS)

    Atassi, M. Zouhair (Inventor); Morrison, Dennis R. (Inventor)

    2009-01-01

    Antibodies have been developed against the different molecular forms of urokinase using synthetic peptides as immunogens. The peptides were synthesized specifically to represent those regions of the urokinase molecules which are exposed in the three-dimensional configuration of the molecule and are uniquely homologous to urokinase. Antibodies are directed against the lysine 158-isoleucine 159 peptide bond which is cleaved during activation from the single-chain (ScuPA) form to the bioactive double chain (54 KDa and 33 KDa) forms of urokinase and against the lysine 135 lysine 136 bond that is cleaved in the process of removing the alpha-chain from the 54 KDa form to produce the 33 KDa form of urokinase. These antibodies enable the direct measurement of the different molecular forms of urokinase from small samples of conditioned medium harvested from cell cultures.

  15. Guinea-pig reaginic antibody

    PubMed Central

    Margni, R. A.; Hajos, Silvia E.

    1973-01-01

    The methods for isolation and purification of a guinea-pig serum protein with homocytotropic antibody activity and characteristics of IgE are described. By precipitation in the equivalence zone or immunoadsorption and chromatography on DEAE-cellulose, we isolated an homocytotropic antibody, that was not able to give a precipitin line when it was reacted directly with the antigen. It was capable of sensitizing guinea-pig skin for PCA after a latent period of 24–48 hours but not after 3 hours; it was sensitive to treatment with mercaptoethanol. It had antigenic determinants present in the other guinea-pig immunoglobulins and particular antigenic determinants. All these properties make us believe that this protein belongs to an immunoglobulin different from γ1 and similar to the reaginic antibody (IgE) described in other species. ImagesFIG. 3FIG. 4FIG. 5 PMID:4126261

  16. Antibodies to watch in 2013

    PubMed Central

    Reichert, Janice M

    2013-01-01

    The transitions of antibody therapeutics to late-stage clinical development, regulatory review and the market are proceeding at a rapid pace in 2013. Since late 2012, two monoclonal antibody (mAb) therapeutics (itolizumab, trastuzumab emtansine) received their first approvals, first marketing applications for three mAbs (vedolizumab, ramucirumab, obinutuzumab) were submitted to regulatory agencies, and five mAbs (brodalumab, MABp1, moxetumomab pasudotox, tildrakizumab, rilotumumab) entered their first Phase 3 studies. The current total of commercially-sponsored antibody therapeutics undergoing evaluation in late-stage studies is 30. Recently announced study results for farletuzumab, naptumomab estafenatox, and tabalumab indicate that clinical endpoints were not met in some Phase 3 studies of these product candidates. PMID:23727858

  17. Communication: Antibody stability and behavior on surfaces

    NASA Astrophysics Data System (ADS)

    Bush, Derek B.; Knotts, Thomas A.

    2015-08-01

    Antibody microarrays have the potential to revolutionize molecular detection in scientific, medical, and other biosensor applications, but their current use is limited because of poor reliability. It is hypothesized that one reason for their poor performance results from strong antibody-surface interactions that destabilize the antibody structure and create steric interference for antigen recognition. Using a recently developed coarse-grain protein-surface model that has been parameterized against experimental data, antibody-surface interactions for two antibody orientations on two types of surfaces have been investigated. The results show that regardless of attachment geometry, antibodies tend to collapse onto hydrophobic surfaces and exhibit lower overall stability compared to antibodies on hydrophilic surfaces or in bulk solution. The results provide an unprecedented view into the dynamics of antibodies on surfaces and offer new insights into the poor performance exhibited by current antibody microarrays.

  18. Communication: Antibody stability and behavior on surfaces.

    PubMed

    Bush, Derek B; Knotts, Thomas A

    2015-08-14

    Antibody microarrays have the potential to revolutionize molecular detection in scientific, medical, and other biosensor applications, but their current use is limited because of poor reliability. It is hypothesized that one reason for their poor performance results from strong antibody-surface interactions that destabilize the antibody structure and create steric interference for antigen recognition. Using a recently developed coarse-grain protein-surface model that has been parameterized against experimental data, antibody-surface interactions for two antibody orientations on two types of surfaces have been investigated. The results show that regardless of attachment geometry, antibodies tend to collapse onto hydrophobic surfaces and exhibit lower overall stability compared to antibodies on hydrophilic surfaces or in bulk solution. The results provide an unprecedented view into the dynamics of antibodies on surfaces and offer new insights into the poor performance exhibited by current antibody microarrays.

  19. Autologous antibodies that bind neuroblastoma cells.

    PubMed

    Sun, Yujing; Sholler, Giselle S; Shukla, Girja S; Pero, Stephanie C; Carman, Chelsea L; Zhao, Ping; Krag, David N

    2015-11-01

    Antibody therapy of neuroblastoma is promising and our goal is to derive antibodies from patients with neuroblastoma for developing new therapeutic antibodies. The feasibility of using residual bone marrow obtained for clinical indications as a source of tumor cells and a source of antibodies was assessed. From marrow samples, neuroblastoma cells were recovered, grown in cell culture and also implanted into mice to create xenografts. Mononuclear cells from the marrow were used as a source to generate phage display antibody libraries and also hybridomas. Growth of neuroblastoma patient cells was possible both in vitro and as xenografts. Antibodies from the phage libraries and from the monoclonal hybridomas bound autologous neuroblastoma cells with some selectivity. It appears feasible to recover neuroblastoma cells from residual marrow specimens and to generate human antibodies that bind autologous neuroblastoma cells. Expansion of this approach is underway to collect more specimens, optimize methods to generate antibodies, and to evaluate the bioactivity of neuroblastoma-binding antibodies.

  20. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V.

    2013-04-09

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.

  1. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V.

    2010-06-22

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.

  2. Antibody profiling sensitivity through increased reporter antibody layering

    DOEpatents

    Apel, William A.; Thompson, Vicki S.

    2017-03-28

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  3. Antibody profiling sensitivity through increased reporter antibody layering

    DOEpatents

    Apel, William A.; Thompson, Vicki S.

    2013-02-26

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  4. Antibody profiling sensitivity through increased reporter antibody layering

    DOEpatents

    Apel, William A.; Thompson, Vicki S

    2010-04-13

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  5. Antibody profiling sensitivity through increased reporter antibody layering

    SciTech Connect

    Apel, William A; Thompson, Vicki S

    2013-02-26

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  6. Monoclonal Antibodies in Diagnosis and Therapy

    NASA Astrophysics Data System (ADS)

    Waldmann, Thomas A.

    1991-06-01

    Monoclonal antibodies have been applied clinically to the diagnosis and therapy of an array of human disorders, including cancer and infectious diseases, and have been used for the modulation of immune responses. Effective therapy using unmodified monoclonal antibodies has, however, been elusive. Recently, monoclonal antibody-mediated therapy has been revolutionized by advances such as the definition of cell-surface structures on abnormal cells as targets for effective monoclonal antibody action, genetic engineering to create less immunogenic and more effective monoclonal antibodies, and the arming of such antibodies with toxins or radionuclides to enhance their effector function.

  7. Tumor detection using radiolabeled monoclonal antibodies

    SciTech Connect

    Moldofsky, P.J.; Powe, J.; Hammond, N.D.

    1987-01-01

    Radioisotope conjugated to monoclonal antibody products has been used for imaging tumors targeted by the antibody. As imaging progresses, new sets of procedural and technical questions arise. In this chapter, we discuss several current problems in imaging tumor with radiolabeled monoclonal antibody. These include (1) methods for selection of specific antibody and, once the particular antibody is selected, which fragment form is to be used; (2) imaging procedures: what are the optimum imaging parameters, such as optimum time for imaging after administration of tracer and considerations regarding background subtraction; and (3) noninvasive quantitative techniques: quantitation of localization of antibody indirectly from quantitative information in the images.100 references.

  8. Mathematical and experimental analyses of antibody transport in hollow-fiber-based specific antibody filters.

    PubMed

    Hout, Mariah S; Federspiel, William J

    2003-01-01

    We are developing hollow fiber-based specific antibody filters (SAFs) that selectively remove antibodies of a given specificity directly from whole blood, without separation of the plasma and cellular blood components and with minimal removal of plasma proteins other than the targeted pathogenic antibodies. A principal goal of our research is to identify the primary mechanisms that control antibody transport within the SAF and to use this information to guide the choice of design and operational parameters that maximize the SAF-based antibody removal rate. In this study, we formulated a simple mathematical model of SAF-based antibody removal and performed in vitro antibody removal experiments to test key predictions of the model. Our model revealed three antibody transport regimes, defined by the magnitude of the Damköhler number Da (characteristic antibody-binding rate/characteristic antibody diffusion rate): reaction-limited (Da /= 10). For a given SAF geometry, blood flow rate, and antibody diffusivity, the highest antibody removal rate was predicted for diffusion-limited antibody transport. Additionally, for diffusion-limited antibody transport the predicted antibody removal rate was independent of the antibody-binding rate and hence was the same for any antibody-antigen system and for any patient within one antibody-antigen system. Using SAF prototypes containing immobilized bovine serum albumin (BSA), we measured anti-BSA removal rates consistent with transport in the intermediate regime (Da approximately 3). We concluded that initial SAF development work should focus on achieving diffusion-limited antibody transport by maximizing the SAF antibody-binding capacity (hence maximizing the characteristic antibody-binding rate). If diffusion-limited antibody transport is achieved, the antibody removal rate may be raised further by increasing the number and length of the SAF fibers and by

  9. Dengue antibodies in blood donors

    PubMed Central

    Ribas-Silva, Rejane Cristina; Eid, Andressa Ahmad

    2012-01-01

    Background Dengue is an urban arbovirus whose etiologic agent is a virus of the genus Flavorius with four distinct antigen serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) that is transmitted to humans through the bite of the mosquito Aedes aegypti. The Campo Mourão region in Brazil is endemic for dengue fever. Obtective The aim of this study was to evaluate the presence of IgG and IgM antibodies specific to the four serotypes of dengue in donors of the blood donor service in the city of Campo Mourão. Methods Epidemiological records were evaluated and 4 mL of peripheral blood from 213 blood donors were collected in tubes without anticoagulant. Serum was then obtained and immunochromatographic tests were undertaken (Imuno-Rápido Dengue IgM/IgGTM). Individuals involved in the study answered a social and epidemiological questionnaire on data which included age, gender and diagnosis of dengue. Results Only three (1.4%) of the 213 blood tests were positive for IgG anti-dengue antibodies. No donors with IgM antibody, which identifies acute infection, were identified. Conclusions The results of the current analysis show that the introduction of quantitative or molecular serological methods to determine the presence of anti-dengue antibodies or the detection of the dengue virus in blood donors in endemic regions should be established so that the quality of blood transfusions is guaranteed. PMID:23049418

  10. Serum Antibody Biomarkers for ASD

    DTIC Science & Technology

    2014-10-01

    Systemic immunologic alterations in autistic individuals often have been associated with autoimmunity; in particular, the generation of antibodies...Molloy et al., 2006; Braunschweig et al., 2013). Systemic immunologic alterations in autistic individuals often have been associated with autoimmunity...Jyonouchi H, Geng L, Ruby A, Zimmerman-Bier B. (2005) Dysregulated innate immune responses in young children with autism spectrum disorders: their

  11. Antibody Isotype Switching in Vertebrates.

    PubMed

    Senger, Kate; Hackney, Jason; Payandeh, Jian; Zarrin, Ali A

    2015-01-01

    The humoral or antibody-mediated immune response in vertebrates has evolved to respond to diverse antigenic challenges in various anatomical locations. Diversification of the immunoglobulin heavy chain (IgH) constant region via isotype switching allows for remarkable plasticity in the immune response, including versatile tissue distribution, Fc receptor binding, and complement fixation. This enables antibody molecules to exert various biological functions while maintaining antigen-binding specificity. Different immunoglobulin (Ig) classes include IgM, IgD, IgG, IgE, and IgA, which exist as surface-bound and secreted forms. High-affinity autoantibodies are associated with various autoimmune diseases such as lupus and arthritis, while defects in components of isotype switching are associated with infections. A major route of infection used by a large number of pathogens is invasion of mucosal surfaces within the respiratory, digestive, or urinary tract. Most infections of this nature are initially limited by effector mechanisms such as secretory IgA antibodies. Mucosal surfaces have been proposed as a major site for the genesis of adaptive immune responses, not just in fighting infections but also in tolerating commensals and constant dietary antigens. We will discuss the evolution of isotype switching in various species and provide an overview of the function of various isotypes with a focus on IgA, which is universally important in gut homeostasis as well as pathogen clearance. Finally, we will discuss the utility of antibodies as therapeutic modalities.

  12. Rickettsial Antibodies in Multiple Sclerosis

    PubMed Central

    Field, E. J.; Chambers, Mavis

    1970-01-01

    The serum of subjects with multiple sclerosis or other degenerative neurological diseases and that of normal controls does not differ significantly in its content of antibodies to several rickettsial antigens. There is no evidence for a rickettsial aetiology of multiple sclerosis, and no grounds for an extended clinical trial of Le Gac's full method of treatment with antibiotics. PMID:4983591

  13. Antibody Engineering for Pursuing a Healthier Future

    PubMed Central

    Saeed, Abdullah F. U. H.; Wang, Rongzhi; Ling, Sumei; Wang, Shihua

    2017-01-01

    Since the development of antibody-production techniques, a number of immunoglobulins have been developed on a large scale using conventional methods. Hybridoma technology opened a new horizon in the production of antibodies against target antigens of infectious pathogens, malignant diseases including autoimmune disorders, and numerous potent toxins. However, these clinical humanized or chimeric murine antibodies have several limitations and complexities. Therefore, to overcome these difficulties, recent advances in genetic engineering techniques and phage display technique have allowed the production of highly specific recombinant antibodies. These engineered antibodies have been constructed in the hunt for novel therapeutic drugs equipped with enhanced immunoprotective abilities, such as engaging immune effector functions, effective development of fusion proteins, efficient tumor and tissue penetration, and high-affinity antibodies directed against conserved targets. Advanced antibody engineering techniques have extensive applications in the fields of immunology, biotechnology, diagnostics, and therapeutic medicines. However, there is limited knowledge regarding dynamic antibody development approaches. Therefore, this review extends beyond our understanding of conventional polyclonal and monoclonal antibodies. Furthermore, recent advances in antibody engineering techniques together with antibody fragments, display technologies, immunomodulation, and broad applications of antibodies are discussed to enhance innovative antibody production in pursuit of a healthier future for humans. PMID:28400756

  14. Polyclonal and monoclonal antibodies in clinic.

    PubMed

    Wootla, Bharath; Denic, Aleksandar; Rodriguez, Moses

    2014-01-01

    Immunoglobulins (Ig) or antibodies are heavy plasma proteins, with sugar chains added to amino-acid residues by N-linked glycosylation and occasionally by O-linked glycosylation. The versatility of antibodies is demonstrated by the various functions that they mediate such as neutralization, agglutination, fixation with activation of complement and activation of effector cells. Naturally occurring antibodies protect the organism against harmful pathogens, viruses and infections. In addition, almost any organic chemical induces antibody production of antibodies that would bind specifically to the chemical. These antibodies are often produced from multiple B cell clones and referred to as polyclonal antibodies. In recent years, scientists have exploited the highly evolved machinery of the immune system to produce structurally and functionally complex molecules such as antibodies from a single B clone, heralding the era of monoclonal antibodies. Most of the antibodies currently in the clinic, target components of the immune system, are not curative and seek to alleviate symptoms rather than cure disease. Our group used a novel strategy to identify reparative human monoclonal antibodies distinct from conventional antibodies. In this chapter, we discuss the therapeutic relevance of both polyclonal and monoclonal antibodies in clinic.

  15. The European antibody network's practical guide to finding and validating suitable antibodies for research.

    PubMed

    Roncador, Giovanna; Engel, Pablo; Maestre, Lorena; Anderson, Amanda P; Cordell, Jacqueline L; Cragg, Mark S; Šerbec, Vladka Č; Jones, Margaret; Lisnic, Vanda J; Kremer, Leonor; Li, Demin; Koch-Nolte, Friedrich; Pascual, Núria; Rodríguez-Barbosa, Jose-Ignacio; Torensma, Ruurd; Turley, Helen; Pulford, Karen; Banham, Alison H

    2016-01-01

    Antibodies are widely exploited as research/diagnostic tools and therapeutics. Despite providing exciting research opportunities, the multitude of available antibodies also offers a bewildering array of choice. Importantly, not all companies comply with the highest standards, and thus many reagents fail basic validation tests. The responsibility for antibodies being fit for purpose rests, surprisingly, with their user. This paper condenses the extensive experience of the European Monoclonal Antibody Network to help researchers identify antibodies specific for their target antigen. A stepwise strategy is provided for prioritising antibodies and making informed decisions regarding further essential validation requirements. Web-based antibody validation guides provide practical approaches for testing antibody activity and specificity. We aim to enable researchers with little or no prior experience of antibody characterization to understand how to determine the suitability of their antibody for its intended purpose, enabling both time and cost effective generation of high quality antibody-based data fit for publication.

  16. The European antibody network's practical guide to finding and validating suitable antibodies for research

    PubMed Central

    Roncador, Giovanna; Engel, Pablo; Maestre, Lorena; Anderson, Amanda P.; Cordell, Jacqueline L.; Cragg, Mark S.; Šerbec, Vladka Č.; Jones, Margaret; Lisnic, Vanda J.; Kremer, Leonor; Li, Demin; Koch-Nolte, Friedrich; Pascual, Núria; Rodríguez-Barbosa, Jose-Ignacio; Torensma, Ruurd; Turley, Helen; Pulford, Karen; Banham, Alison H.

    2016-01-01

    Antibodies are widely exploited as research/diagnostic tools and therapeutics. Despite providing exciting research opportunities, the multitude of available antibodies also offers a bewildering array of choice. Importantly, not all companies comply with the highest standards, and thus many reagents fail basic validation tests. The responsibility for antibodies being fit for purpose rests, surprisingly, with their user. This paper condenses the extensive experience of the European Monoclonal Antibody Network to help researchers identify antibodies specific for their target antigen. A stepwise strategy is provided for prioritising antibodies and making informed decisions regarding further essential validation requirements. Web-based antibody validation guides provide practical approaches for testing antibody activity and specificity. We aim to enable researchers with little or no prior experience of antibody characterization to understand how to determine the suitability of their antibody for its intended purpose, enabling both time and cost effective generation of high quality antibody-based data fit for publication. PMID:26418356

  17. Anti-Sulfoglucuronosyl Paragloboside Antibody

    PubMed Central

    Li, Dongpei; Usuki, Seigo; Quarles, Brandy; Rivner, Michael H.; Ariga, Toshio

    2016-01-01

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons. Although the etiology of ALS is obscure, genetic studies of familiar ALS suggest a multifactorial etiology for this condition. Similarly, there probably are multiple causes for sporadic ALS. Autoimmune-mediated motor neuron dysfunction is one proposed etiology for sporadic ALS. In the present study, anti-glycolipid antibodies including GM1, GD1b, GD3, and sulfoglucuronosyl paragloboside (SGPG) were investigated in the sera of a large number of patient samples, including 113 ALS patients and 50 healthy controls, by means of enzyme-linked immunosorbent assay with affinity parametric complex criterion evaluation and thin-layer chromatography immunooverlay (immuno-TLC). Anti-SGPG antibodies were found in the sera of 13.3% ALS patients (15 out of 113). The highest titer reached 1:1600. The presence of anti-SGPG antibodies in the serum samples was also confirmed by immuno-TLC. Importantly, a multiple logistic regression analysis showed that the presence of anti-SGPG antibody was positively correlated with age (p < .01) and negatively correlated with ALS Functional Rating Scale score (p < .05). Moreover, the localization of SGPG-immunoreactivity on the motor neurons of rat spinal cord and a mouse motor neuronal cell line, NSC-34 was observed by an immunofluorescence method. These data suggest that SGPG could represent a specific pathogenic antigen in those ALS patients. The presence of anti-SGPG antibodies in the serum of ALS patients should represent a diagnostic biomarker of ALS, and it could reflect the severity of the disease. PMID:27683876

  18. Encephalitis and AMPA receptor antibodies

    PubMed Central

    Höftberger, Romana; van Sonderen, Agnes; Leypoldt, Frank; Houghton, David; Geschwind, Michael; Gelfand, Jeffrey; Paredes, Mercedes; Sabater, Lidia; Saiz, Albert; Titulaer, Maarten J.; Graus, Francesc

    2015-01-01

    Objective: We report the clinical features, comorbidities, and outcome of 22 newly identified patients with antibodies to the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR). Methods: This was a retrospective review of patients diagnosed between May 2009 and March 2014. Immunologic techniques have been reported previously. Results: Patients' median age was 62 years (range 23–81; 14 female). Four syndromes were identified: 12 (55%) patients presented with distinctive limbic encephalitis (LE), 8 (36%) with limbic dysfunction along with multifocal/diffuse encephalopathy, one with LE preceded by motor deficits, and one with psychosis with bipolar features. Fourteen patients (64%) had a tumor demonstrated pathologically (5 lung, 4 thymoma, 2 breast, 2 ovarian teratoma) or radiologically (1 lung). Additional antibodies occurred in 7 patients (3 onconeuronal, 1 tumor-related, 2 cell surface, and 1 tumor-related and cell surface), all with neurologic symptoms or tumor reflecting the concurrent autoimmunity. Treatment and outcome were available from 21 patients (median follow-up 72 weeks, range 5–266): 5 had good response to immunotherapy and tumor therapy, 10 partial response, and 6 did not improve. Eventually 5 patients died; all had a tumor or additional paraneoplastic symptoms related to onconeuronal antibodies. Coexistence of onconeuronal antibodies predicted a poor outcome (p = 0.009). Conclusion: Anti-AMPAR encephalitis usually manifests as LE, can present with other symptoms or psychosis, and is paraneoplastic in 64% of cases. Complete and impressive neurologic improvement can occur, but most patients have partial recovery. Screening for a tumor and onconeuronal antibodies is important because their detection influences outcome. PMID:25979696

  19. How Is Antiphospholipid Antibody Syndrome Treated?

    MedlinePlus

    ... Is Antiphospholipid Antibody Syndrome Treated? Antiphospholipid antibody syndrome (APS) has no cure. However, medicines can help prevent ... existing clots from getting larger. You may have APS and another autoimmune disorder, such as lupus . If ...

  20. Indirect immunofluorescent antibody test in chicken leucocytozoonosis.

    PubMed

    Isobe, T; Akiba, K

    1982-01-01

    The indirect immunofluorescent antibody technique was applied to detection of antigens of different developmental stages of Leucocytozoon caulleryi and antibodies in the sera of chickens infected with L. caulleryi by using second generation merozoite and gametocyte antigens. Zygotes, ookinetes and sporozoites in midges, and second generation merozoites and gametocytes in chickens indicated specific fluorescence. Indirect immunofluorescent antibody titers were higher than agar gel precipitation antibody titers. So the detection of the antibody by the indirect immunofluorescent antibody technique was possible in the sera in which the antibody could not be detected by the agal gel precipitation test. Therefore, the indirect immunofluorescent antibody technique was applicable to chicken leucocytozoonosis as a highly sensitive serological diagnostic method.

  1. ANCA / MPO / PR3 Antibodies Test

    MedlinePlus

    ... may be ordered with a test for anti- Saccharomyces cerevisiae antibodies (ASCA) when a person has signs and ... If atypical ANCA is positive and ASCA (anti- Saccharomyces cerevisiae antibodies) is negative, then it is likely that ...

  2. Detection of Campylobacter species using monoclonal antibodies

    NASA Astrophysics Data System (ADS)

    Young, Colin R.; Lee, Alice; Stanker, Larry H.

    1999-01-01

    A panel of species specific monoclonal antibodies were raised to Campylobacter coli, Campylobacter jejuni and Campylobacter lari. The isotypes, and cross-reactivity profiles of each monoclonal antibody against an extensive panel of micro- organisms, were determined.

  3. Antibody reactivity with Skinner HSV vaccine.

    PubMed

    Muniu, E M; Durham, J; Shariff, D; Hartley, C E; Fuller, A; Melling, J; Wiblin, C; Wilkins, G; Buchan, A; Skinner, G R

    1987-01-01

    Antibody reactivity against herpes simplex virus (HSV) was investigated in 15 subjects who received three subcutaneous immunisations with Skinner HSV vaccine. Humoral antibody responses were detected against type 1 HSV in every subject and against type 2 HSV in all but one subject; immuno-precipitating antibody responses were infrequently detected. There was no antibody reactivity against host-cell (MRC-5), foetal calf serum or rubella virus antigen. None of the vaccinated subjects developed clinical evidence of herpes genitalis.

  4. Anti-DNA antibodies in SLE

    SciTech Connect

    Voss, E.W.

    1988-01-01

    This book contains 8 chapters. Some of the titles are: Anti-DNA Antibodies in SLE: Historical Perspective; Specificity of Anti-DNA Antibodies in Systemic Lupus Erythematosus; Monoclonial Autoimmune Anti-DNA Antibodies; and Structure--Function Analyses of Anti-DNA Autoantibodies.

  5. Application of monoclonal antibodies in tumor pathology

    SciTech Connect

    Ruiter, D.J. ); Fleuren, G.J.; Warnaar, S.O. )

    1987-01-01

    This book contains papers under the following three section headings: Basic and technical aspects; Tumor associated antigens; and Practical application and case presentations. Some of the paper titles are: Monoclonal antibodies to oncofetal antigens; Monoclonal antibodies against ovarian cancer; and Tumor associated antigens and oncogene products defined by monoclonal antibodies.

  6. Antidisialosyl antibodies in chronic idiopathic ataxic neuropathy.

    PubMed

    Serrano-Munuera, C; Rojas-García, R; Gallardo, E; De Luna, N; Buenaventura, I; Ferrero, M; García, T; García-Merino, J A; González-Rodríguez, C; Guerriero, A; Marco, M; Márquez, C; Grau, J M; Graus, F; Illa, I

    2002-11-01

    Antidisialosyl antibodies were found in two out of 13 patients with chronic idiopathic ataxic neuropathy (CIAN) and not in 32 patients with different sensory neuropathies of known cause. This finding confirms the association of antidisialosyl antibodies and CIAN regardless of the absence of the M band. These antibodies may have pathogenic relevance; however, larger series are needed to establish their clinical significance.

  7. Effects of medium concentration on antibody production

    NASA Technical Reports Server (NTRS)

    Williams, J.

    1984-01-01

    Antibody production by two different cell lines was measured as the media were supplemented with varied amounts of glucose and fetal bovine serum. Both cell lines elaborated antidinitrophenyl hapten antibodies. Two basic media were used: RPMI 1640 and Dulbecco's modified Eagle's medium. The production of antibodies was followed from 0 to 180 h and was assayed by radioimmunoassay.

  8. Effects of medium concentration on antibody production

    NASA Technical Reports Server (NTRS)

    Williams, J.

    1984-01-01

    Antibody production by two different cell lines was measured as the media were supplemented with varied amounts of glucose and fetal bovine serum. Both cell lines elaborated antidinitrophenyl hapten antibodies. Two basic media were used: RPMI 1640 and Dulbecco's modified Eagle's medium. The production of antibodies was followed from 0 to 180 h and was assayed by radioimmunoassay.

  9. Antibody biosimilars: Fears or opportunities?

    PubMed Central

    Guillon-Munos, Audrey; Daguet, Arnaud; Watier, Hervé

    2014-01-01

    The annual “LabEx MAbImprove industrial workshops” are primarily intended to provide scientists involved in therapeutic antibodies, a comprehensive view about topics of interest for the pharmaceutical industry. They are organized by the “LabEx MAbImprove industrial committee”, for this first edition especially in partnership with ARITT, the regional agency for innovation and technology transfer which operates in the French Région Centre, the 1st French region for pharmaceutical production. The 2013 edition, held May 28 at the Vinci Center of Tours, was dedicated to antibody biosimilars. Depending on opinions, the impending expiry of antibody patents and the imminent marketing approval of competitors to blockbusters can be perceived as good or bad things. Fears or opportunities? Risks for patients? Breath of fresh air for the health systems? Opportunity for re-industrializing France? In this context, it is necessary for people to form a fair and informed opinion on the current landscape of antibody biosimilars. In particular, this is especially important for scientists from the academic world, from the industry or from the regulation agencies, for pharmacists, for pharmacovigilance specialists, for health authorities, and staff from health insurance and decision makers. The first session was devoted to market and regulatory issues, and included both an overview of the evolution of the patent landscape and a description of biosimilars regulation in the European Union (EU). This session was closed by a talk on manufacturing processes for biosimilars. In the next session, quality control attributes of biosimilars were discussed and compared with the consistent quality of biotechnology products to raise the question: “How close is close enough?” In vitro assays for evaluating the Fc function of therapeutic antibodies were also discussed. The third session focused on development of biosimilars and primarily on the stepwise process for introducing an antibody

  10. Recombinant antibodies and their use in biosensors.

    PubMed

    Zeng, Xiangqun; Shen, Zhihong; Mernaugh, Ray

    2012-04-01

    Inexpensive, noninvasive immunoassays can be used to quickly detect disease in humans. Immunoassay sensitivity and specificity are decidedly dependent upon high-affinity, antigen-specific antibodies. Antibodies are produced biologically. As such, antibody quality and suitability for use in immunoassays cannot be readily determined or controlled by human intervention. However, the process through which high-quality antibodies can be obtained has been shortened and streamlined by use of genetic engineering and recombinant antibody techniques. Antibodies that traditionally take several months or more to produce when animals are used can now be developed in a few weeks as recombinant antibodies produced in bacteria, yeast, or other cell types. Typically most immunoassays use two or more antibodies or antibody fragments to detect antigens that are indicators of disease. However, a label-free biosensor, for example, a quartz-crystal microbalance (QCM) needs one antibody only. As such, the cost and time needed to design and develop an immunoassay can be substantially reduced if recombinant antibodies and biosensors are used rather than traditional antibody and assay (e.g. enzyme-linked immunosorbant assay, ELISA) methods. Unlike traditional antibodies, recombinant antibodies can be genetically engineered to self-assemble on biosensor surfaces, at high density, and correctly oriented to enhance antigen-binding activity and to increase assay sensitivity, specificity, and stability. Additionally, biosensor surface chemistry and physical and electronic properties can be modified to further increase immunoassay performance above and beyond that obtained by use of traditional methods. This review describes some of the techniques investigators have used to develop highly specific and sensitive, recombinant antibody-based biosensors for detection of antigens in simple or complex biological samples.

  11. Antibody Repertoire Development in Swine.

    PubMed

    Butler, J E; Wertz, Nancy; Sinkora, Marek

    2017-02-08

    We describe the domestication of the species, explore its value to agriculture and bioscience, and compare its immunoglobulin (Ig) genes to those of other vertebrates. For encyclopedic information, we cite earlier reviews and chapters. We provide current gene maps for the heavy and light chain loci and describe their polygeny and polymorphy. B-cell and antibody repertoire development is a major focus, and we present findings that challenge several mouse-centric paradigms. We focus special attention on the role of ileal Peyer's patches, the largest secondary lymphoid tissues in newborn piglets and a feature of all artiodactyls. We believe swine fetal development and early class switch evolved to provide natural secretory IgA antibodies able to prevent translocation of bacteria from the gut while the bacterial PAMPs drive development of adaptive immunity. We discuss the value of using the isolator piglet model to address these issues.

  12. Expression studies of catalytic antibodies

    SciTech Connect

    Ulrich, H.D.; Patten, P.A.; Yang, P.L.

    1995-12-05

    We have examined the positive influence of human constant regions on the folding and bacterial expression of active soluble mouse immunoglobulin variable domains derived form a number of catalytic antibodies. Expression yields of eight hybridoma-and myeloma-derived chimeric Fab fragments are compared in both shake flasks and high-density fermentation. In addition the usefulness of this system for the generation of in vivo expression libraries is examined by constructing and expressing combinations of heavy and light chain variable regions that were not selected as a pair during an immune response. A mutagenesis study of one of the recombinant catalytic Fab fragments reveals that single amino acid substitutions can have dramatic effects on the expression yield. This system should be generally applicable to the production of Fab fragments of catalytic and other hybridoma-derived antibodies for crystallographic and structure-function studies. 41 refs., 4 figs., 1 tab.

  13. Autoimmunity in Primary Antibody Deficiencies.

    PubMed

    Azizi, Gholamreza; Ahmadi, Moslem; Abolhassani, Hassan; Yazdani, Reza; Mohammadi, Hamed; Mirshafiey, Abbas; Rezaei, Nima; Aghamohammadi, Asghar

    2016-01-01

    Primary antibody deficiencies (PADs) are the most common inherited primary immunodeficiencies in humans, characterized by hypogammaglobulinemia, an inability to produce specific antibodies, and recurrent infections mainly caused by encapsulated bacteria. However, it has been shown that inflammatory disorders, granulomatous lesions, lymphoproliferative diseases, cancer, and autoimmunity are associated with the various types of PAD. Both systemic and organ-specific autoimmune diseases could be attributed to B-cell defects in PAD patients. Immune thrombocytopenic purpura and autoimmune hemolytic anemia are the most common autoimmune disorders in this group of patients. The aim of this review is to describe the proposed mechanisms for autoimmunity and to review the literature with respect to the reported autoimmune disorders in each type of PAD. © 2016 S. Karger AG, Basel.

  14. Antibody engineering and therapeutics conference

    PubMed Central

    Larrick, James W; Parren, Paul WHI; Huston, James S; Plückthun, Andreas; Bradbury, Andrew; Tomlinson, Ian M; Chester, Kerry A; Burton, Dennis R; Adams, Gregory P; Weiner, Louis M; Scott, Jamie K; Alfenito, Mark R; Veldman, Trudi; Reichert, Janice M

    2014-01-01

    The 25th anniversary of the Antibody Engineering & Therapeutics Conference, the Annual Meeting of The Antibody Society, will be held in Huntington Beach, CA, December 7–11, 2014. Organized by IBC Life Sciences, the event will celebrate past successes, educate participants on current activities and offer a vision of future progress in the field. Keynote addresses will be given by academic and industry experts Douglas Lauffenburger (Massachusetts Institute of Technology), Ira Pastan (National Cancer Institute), James Wells (University of California, San Francisco), Ian Tomlinson (GlaxoSmithKline) and Anthony Rees (Rees Consulting AB and Emeritus Professor, University of Bath). These speakers will provide updates of their work, placed in the context of the substantial growth of the industry over the past 25 years. PMID:25517297

  15. Viral Epitopes and Monoclonal Antibodies: Isolation of Blocking Antibodies that Inhibit Virus Neutralization

    NASA Astrophysics Data System (ADS)

    Massey, Richard J.; Schochetman, Gerald

    1981-07-01

    The inability of pathogenic animal viruses to be completely neutralized by antibodies can lead to chronic viral infections in which infectious virus persists even in the presence of excess neutralizing antibody. A mechanism that results in this nonneutralized fraction of virus was defined by the topographical relationships of viral epitopes identified with monoclonal antibodies wherein monoclonal antibodies bind to virus and sterically block the binding of neutralizing antibodies.

  16. Phenotypic screening: the future of antibody discovery.

    PubMed

    Gonzalez-Munoz, Andrea L; Minter, Ralph R; Rust, Steven J

    2016-01-01

    Most antibody therapeutics have been isolated from high throughput target-based screening. However, as the number of validated targets diminishes and the target space becomes increasingly competitive, alternative strategies, such as phenotypic screening, are gaining momentum. Here, we review successful phenotypic screens, including those used to isolate antibodies against cancer and infectious agents. We also consider exciting advances in the expression and phenotypic screening of antibody repertoires in single cell autocrine systems. As technologies continue to develop, we believe that antibody phenotypic screening will increase further in popularity and has the potential to provide the next generation of therapeutic antibodies.

  17. Production of Monoclonal Antibody against Human Nestin.

    PubMed

    Hadavi, Reza; Zarnani, Amir Hassan; Ahmadvand, Negah; Mahmoudi, Ahmad Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Sadeghi, Mohammad-Reza; Soltanghoraee, Haleh; Akhondi, Mohammad Mehdi; Tarahomi, Majid; Jeddi-Tehrani, Mahmood; Rabbani, Hodjattallah

    2010-04-01

    We have employed a peptide-based antibody generation protocol for producing antibody against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N- or O-glyco-sylation sequences, we generated a mouse monoclonal antibody capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140-250 kDa was detected by this antibody. This antibody is highly specific and functional in applications such as ELISA, flow cytometry, immunocytochemistry, and Western blot assays.

  18. Production of Monoclonal Antibody against Human Nestin

    PubMed Central

    Hadavi, Reza; Zarnani, Amir Hassan; Ahmadvand, Negah; Mahmoudi, Ahmad Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Sadeghi, Mohammad-Reza; Soltanghoraee, Haleh; Akhondi, Mohammad Mehdi; Tarahomi, Majid; Jeddi-Tehrani, Mahmood; Rabbani, Hodjattallah

    2010-01-01

    We have employed a peptide-based antibody generation protocol for producing antibody against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N- or O-glyco-sylation sequences, we generated a mouse monoclonal antibody capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140–250 kDa was detected by this antibody. This antibody is highly specific and functional in applications such as ELISA, flow cytometry, immunocytochemistry, and Western blot assays. PMID:23407796

  19. Single-Chain Antibody Library

    DOE Data Explorer

    Baird, Cheryl

    Researchers at Pacific Northwest National Laboratory (PNNL) have constructed a nonimmune library consisting of 109 human antibody scFv fragments, which have been cloned and expressed on the surface of yeast. Nanomolar-affinity scFvs are routinely obtained by magnetic bead screening and flow cytometric sorting. The yeast library can be amplified 1010 fold without measurable loss of clonal diversity. This allows for indefinite expansion of the library. All scFv clones can be assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps. The ability to use multiplex library screening demonstrates the utility of this approach for high-throughput antibody isolation for proteomic applications. The yeast library may be used for research projects or teaching performed for U.S. Government purposes only. If you would like to request an aliquot of the single-chain antibody library for your research, please print and fill out the Materials Transfer Agreement (MTA) [PDF, 20K]. The website provides the contact information for mailing the MTA. [copied from http://www.sysbio.org/dataresources/singlechain.stm

  20. RABBIT ANTIBODIES TO STREPTOCOCCAL CARBOHYDRATES

    PubMed Central

    Braun, Dietmar G.; Eichmann, Klaus; Krause, Richard M.

    1969-01-01

    In a search for possible genetic factors which may influence the immune response to the streptococcal carbohydrates, over 100 rabbits have been immunized with streptococcal vaccines, and representative examples of high and low response pairs mated. The concentration of precipitins to the group—specific carbohydrates has been measured in the antisera following primary intravenous immunization with heat-killed streptococcal vaccines, Group A, Group A-variant, and Group C. For the majority of rabbits, the concentration of precipitins varied between 1 and 10 mg/ml of antiserum; while in the minority, it was between 11 and 32 mg/ml. The offspring of rabbits with high antibody levels had a significantly higher concentration of antibody than was seen in the offspring of rabbits of low response parents. Such data suggest that the magnitude of the immune response to these carbohydrate antigens is under some form of genetic control. Not uncommonly in rabbits with hyper-γ-globulinemia following primary immunization, the group-specific precipitins are the predominant component of the γ-globulin. An unusual feature of such components is that they are electrophoretically monodisperse, and possess individual antigenic specificity. In this respect they resemble the myeloma proteins. When a response of this sort is not seen after primary immunization, it may occur after secondary immunization. Therefore, prior exposure to the same or closely related antigen may also have an influence on the occurrence of high concentrations of such uniform antibodies. PMID:5766948

  1. Improved monoclonal antibodies to halodeoxyuridine

    DOEpatents

    Vanderlaan, M.; Dolbeare, F.A.; Gray, J.W.; Thomas, C.B.

    1983-10-18

    The development, method of production, characterization and methods of use of two hybridomas, CIdU-1 (ATCC Accession No. HB-8321) and CIdU-2 (ATCC Accession No. HB-8320), are described. These secrete IgG/sub 1/(K) immunoglobulins that react with halodeoxyuridine (HdU or halodU) such as bromo, chloro, fluoro and iodo deoxyuridine (BrdU, CldU, FdU and IdU), whether these are free in solution or incorporated into single stranded DNA in whole cells. The antibodies do not react with naturally occurring free nucleic acids or with deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) polymers. These antibodies are suitable for use in enzyme immunoassays for free CldU, FdU, IdU and BrdU and for detecting cells with these nucleotides incorporated into them. The monoclonal antibodies are useful in the detection of the sensitivity of tumor cells to specific chemotherapeutic agents, in the measurement of the rate of cellular DNA synthesis, in the measurement of the rate of proliferation of normal and malignant cells and in the detection of HPRT deficiency in cells. 1 tab.

  2. [Molecular mechanisms of antibody synthesis].

    PubMed

    Bartram, C R; Kleihauer, E

    1984-10-01

    Antibodies or immunoglobulins play a central part in the immune system. The basic unit of an antibody is composed of two identical light and two identical heavy chains; each chain contains two functionally and structurally distinct regions: an amino-terminal variable or antigen-binding site, and a carboxy-terminal constant region responsible for immunological effector functions. Thanks to recombinant DNA technology the paradox of a limited number of genes and a virtually unlimited capacity to generate specific antibodies has now been resolved at least in outline. Immunoglobulin chains are encoded in multiple gene segments of three unlinked gene families scattered along chromosomes 2 (kappa light chain), 14 (heavy chain) and 22 (lambda light chain). During B-cell differentiation these genes are assembled by somatic recombination mechanisms to form active genes. The enormous diversity generated by means of DNA rearrangements is supplemented by mutations somatically introduced into variable region sequences. The medical impact of these discoveries will be substantial. Possible applications include identification of B-cell precursors lacking conventional markes, a molecular classification of lymphomas and a precise distinction between monoclonal and polyclonal lymphoproliferative disorders.

  3. Production of therapeutic antibodies with controlled fucosylation.

    PubMed

    Yamane-Ohnuki, Naoko; Satoh, Mitsuo

    2009-01-01

    The clinical success of therapeutic antibodies is demonstrated by the number of antibody therapeutics that have been brought to market and the increasing number of therapeutic antibodies in development. Recombinant antibodies are molecular-targeted therapeutic agents and represent a major new class of drugs. However, it is still very important to optimize and maximize the clinical efficacy of therapeutic antibodies, in part to help lower the cost of therapeutic antibodies by potentially reducing the dose or the duration of treatment. Clinical trials using therapeutic antibodies fully lacking core fucose residue in the Fc oligosaccharides are currently underway, and their remarkable physiological activities in humans in vivo have attracted attention as next-generation therapeutic antibody approaches with improved efficacy. Thus, an industrially applicable antibody production process that provides consistent yields of fully non-fucosylated antibody therapeutics with fixed quality has become a key goal in the successful development of next-generation therapeutic agents. In this article, we review the current technologies for production of therapeutic antibodies with control of fucosylation of the Fc N-glycans.

  4. Broadly Neutralizing Antibodies for HIV Eradication.

    PubMed

    Stephenson, Kathryn E; Barouch, Dan H

    2016-02-01

    Passive transfer of antibodies has long been considered a potential treatment modality for infectious diseases, including HIV. Early efforts to use antibodies to suppress HIV replication, however, were largely unsuccessful, as the antibodies that were studied neutralized only a relatively narrow spectrum of viral strains and were not very potent. Recent advances have led to the discovery of a large portfolio of human monoclonal antibodies that are broadly neutralizing across many HIV-1 subtypes and are also substantially more potent. These antibodies target multiple different epitopes on the HIV envelope, thus allowing for the development of antibody combinations. In this review, we discuss the application of broadly neutralizing antibodies (bNAbs) for HIV treatment and HIV eradication strategies. We highlight bNAbs that target key epitopes, such as the CD4 binding site and the V2/V3-glycan-dependent sites, and we discuss several bNAbs that are currently in the clinical development pipeline.

  5. Methods of identification employing antibody profiles

    DOEpatents

    Francoeur, Ann-Michele

    1993-12-14

    An identification method, applicable to the identification of animals or inanimate objects, is described. The method takes advantage of the set of individual-specific antibodies that are part of the unique antibody repertoire present in animals, by reacting an effective amount of such antibodies with a particular panel, of n-dimensional array (where n is typically one or two) consisting of an effective amount of many different antigens (typically greater than one thousand), to give antibody-antigen complexes. The profile or pattern formed by the antigen-antibody complexes, termed an antibody fingerprint, when revealed by an effective amount of an appropriate detector molecule, is uniquely representative of a particular individual. The method can similarly be used to distinguish genetically, or otherwise similar individuals, or their body parts containing individual-specific antibodies.

  6. Antibodies directed against receptor tyrosine kinases

    PubMed Central

    FAUVEL, Bénédicte; Yasri, Aziz

    2014-01-01

    Approximately 30 therapeutic monoclonal antibodies have already been approved for cancers and inflammatory diseases, and monoclonal antibodies continue to be one of the fastest growing classes of therapeutic molecules. Because aberrant signaling by receptor tyrosine kinases (RTKs) is a commonly observed factor in cancer, most of the subclasses of RTKs are being extensively studied as potential targets for treating malignancies. The first two RTKs that have been targeted by antibody therapy, with five currently marketed antibodies, are the growth factor receptors EGFR and HER2. However, due to systemic side effects, refractory patients and the development of drug resistance, these treatments are being challenged by emerging therapeutics. This review examines current monoclonal antibody therapies against RTKs. After an analysis of agents that have already been approved, we present an analysis of antibodies in clinical development that target RTKs. Finally, we highlight promising RTKs that are emerging as new oncological targets for antibody-based therapy. PMID:24859229

  7. Antiangiogenic antibody improves melanoma detection by fluorescently labeled therapeutic antibodies.

    PubMed

    Sweeny, Larissa; Prince, Andrew; Patel, Neel; Moore, Lindsay S; Rosenthal, Eben L; Hughley, Brian B; Warram, Jason M

    2016-12-01

    Evaluate if vascular normalization with an antiangiogenic monoclonal antibody improves detection of melanoma using fluorescently labeled antibody-based imaging. Preclinical. Panitumumab and control IgG were covalently linked to a near-infrared fluorescent probe (IRDye800CW). Immunodeficient mice with ear xenografts of melanoma cell lines (A375 and SKMEL5) were systemically injected (200 μg, tail vein) with either IgG-IRDye800CW, panitumumab-IRDye800CW, or a combination (bevacizumab [5mg/kg], administered 72 hours prepanitumumab-IRDye800CW) (n = 5). Primary tumors were imaged with open-field (LUNA, Novadaq, Toronto, Ontario, Canada) and closed-field (Pearl, LI-COR Biosciences, Lincoln, NB) imaging devices. Postresection, the concentration of labeled antibody within the tumor (μg/g) was calculated using normalized standards. The mean fluorescence within the melanoma tumors was greater for the combination group compared to panitumumab alone for both cell lines (P < 0.001). The tumor-to-background ratio (TBR) for the A375 tumors was greater for the combination (3.4-7.1) compared to the panitumumab alone (3.2-5.0) (P = 0.04). The TBR for SKMEL5 tumors was greater for the combination (2.4-6.0) compared to the panitumumab alone (2.2-3.9) (P = 0.02). Within A375 tumors, the concentration was lower for panitumumab (0.51 μg/g) compared to combination group (0.68 μg/g) (P = 0.036). Within SKMEL5 tumors, the concentration was lower for panitumumab (0.0.17 μg/g) compared to combination group (0.35 μg/g) (P = 0.048). Residual tumor (1.0-0.2 mg) could be differentiated from background in both panitumumab and combination groups. For both cell lines, panitumumab and combination groups had greater mean fluorescence of the tumor compared to control IgG. The addition of antiangiogenic therapy improves uptake of fluorescently labeled monoclonal antibodies within melanoma tumors. Clinical translation could improve detection of melanoma intraoperatively, reducing positive margins

  8. Solid-phase antibody capture hemadsorption assay for detection of hepatitis A virus immunoglobulin M antibodies.

    PubMed Central

    Summers, P L; Dubois, D R; Cohen, W H; Macarthy, P O; Binn, L N; Sjogren, M H; Snitbhan, R; Innis, B L; Eckels, K H

    1993-01-01

    A solid-phase antibody capture hemadsorption (SPACH) assay was developed to detect hepatitis A virus (HAV)-specific immunoglobulin M (IgM) antibodies in sera from humans recently infected with hepatitis. The assay is performed with microtiter plates coated with anti-human IgM antibodies to capture IgM antibodies from the test sera. HAV-specific IgM antibody is detected by the addition of HAV hemagglutinating antigen and goose erythrocytes. Hemadsorption of erythrocytes to antigen-antibody complexes attached to the solid phase indicate the presence of IgM antibodies. The SPACH assay was compared to a commercial radioimmunoassay and was found to be equally or more sensitive and specific for the detection of HAV IgM antibodies. The SPACH assay is an alternative, rapid assay that doesn't require hazardous substrates or radioactivity for the detection of HAV-specific antibodies. PMID:8388890

  9. Generation of monospecific antibodies based on affinity capture of polyclonal antibodies

    PubMed Central

    Hjelm, Barbara; Forsström, Björn; Igel, Ulrika; Johannesson, Henrik; Stadler, Charlotte; Lundberg, Emma; Ponten, Fredrik; Sjöberg, Anna; Rockberg, Johan; Schwenk, Jochen M; Nilsson, Peter; Johansson, Christine; Uhlén, Mathias

    2011-01-01

    A method is described to generate and validate antibodies based on mapping the linear epitopes of a polyclonal antibody followed by sequential epitope-specific capture using synthetic peptides. Polyclonal antibodies directed towards four proteins RBM3, SATB2, ANLN, and CNDP1, potentially involved in human cancers, were selected and antibodies to several non-overlapping epitopes were generated and subsequently validated by Western blot, immunohistochemistry, and immunofluorescence. For all four proteins, a dramatic difference in functionality could be observed for these monospecific antibodies directed to the different epitopes. In each case, at least one antibody was obtained with full functionality across all applications, while other epitope-specific fractions showed no or little functionality. These results present a path forward to use the mapped binding sites of polyclonal antibodies to generate epitope-specific antibodies, providing an attractive approach for large-scale efforts to characterize the human proteome by antibodies. PMID:21898641

  10. Distinction between MOG antibody-positive and AQP4 antibody-positive NMO spectrum disorders

    PubMed Central

    Sato, Douglas Kazutoshi; Callegaro, Dagoberto; Lana-Peixoto, Marco Aurelio; Waters, Patrick J.; Jorge, Frederico M. de Haidar; Takahashi, Toshiyuki; Nakashima, Ichiro; Apostolos-Pereira, Samira Luisa; Talim, Natalia; Simm, Renata Faria; Lino, Angelina Maria Martins; Misu, Tatsuro; Leite, Maria Isabel; Aoki, Masashi

    2014-01-01

    Objective: To evaluate clinical features among patients with neuromyelitis optica spectrum disorders (NMOSD) who have myelin oligodendrocyte glycoprotein (MOG) antibodies, aquaporin-4 (AQP4) antibodies, or seronegativity for both antibodies. Methods: Sera from patients diagnosed with NMOSD in 1 of 3 centers (2 sites in Brazil and 1 site in Japan) were tested for MOG and AQP4 antibodies using cell-based assays with live transfected cells. Results: Among the 215 patients with NMOSD, 7.4% (16/215) were positive for MOG antibodies and 64.7% (139/215) were positive for AQP4 antibodies. No patients were positive for both antibodies. Patients with MOG antibodies represented 21.1% (16/76) of the patients negative for AQP4 antibodies. Compared with patients with AQP4 antibodies or patients who were seronegative, patients with MOG antibodies were more frequently male, had a more restricted phenotype (optic nerve more than spinal cord), more frequently had bilateral simultaneous optic neuritis, more often had a single attack, had spinal cord lesions distributed in the lower portion of the spinal cord, and usually demonstrated better functional recovery after an attack. Conclusions: Patients with NMOSD with MOG antibodies have distinct clinical features, fewer attacks, and better recovery than patients with AQP4 antibodies or patients seronegative for both antibodies. PMID:24415568

  11. Antibodies to human fetal erythroid cells from a nonimmune phage antibody library

    PubMed Central

    Huie, Michael A.; Cheung, Mei-Chi; Muench, Marcus O.; Becerril, Baltazar; Kan, Yuet W.; Marks, James D.

    2001-01-01

    The ability to isolate fetal nucleated red blood cells (NRBCs) from the maternal circulation makes possible prenatal genetic analysis without the need for diagnostic procedures that are invasive for the fetus. Such isolation requires antibodies specific to fetal NRBCs. To generate a panel of antibodies to antigens present on fetal NRBCs, a new type of nonimmune phage antibody library was generated in which multiple copies of antibody fragments are displayed on each phage. Antibody fragments specific for fetal NRBCs were isolated by extensive predepletion of the phage library on adult RBCs and white blood cells (WBCs) followed by positive selection and amplification on fetal liver erythroid cells. After two rounds of selection, 44% of the antibodies analyzed bound fetal NRBCs, with two-thirds of these showing no binding of WBCs. DNA fingerprint analysis revealed the presence of at least 16 unique antibodies. Antibody specificity was confirmed by flow cytometry, immunohistochemistry, and immunofluorescence of total fetal liver and adult RBCs and WBCs. Antibody profiling suggested the generation of antibodies to previously unknown fetal RBC antigens. We conclude that multivalent display of antibodies on phage leads to efficient selection of panels of specific antibodies to cell surface antigens. The antibodies generated to fetal RBC antigens may have clinical utility for isolating fetal NRBCs from maternal circulation for noninvasive prenatal genetic diagnosis. Some of the antibodies may also have possible therapeutic utility for erythroleukemia. PMID:11226299

  12. Individual-specific antibody identification methods

    DOEpatents

    Francoeur, Ann -Michele

    1989-11-14

    An identification method, applicable to the identification of animals or inanimate objects, is described. The method takes advantage of a hithertofore unknown set of individual-specific, or IS antibodies, that are part of the unique antibody repertoire present in animals, by reacting an effective amount of IS antibodies with a particular panel, or n-dimensional array (where n is typically one or two) consisting of an effective amount of many different antigens (typically greater than one thousand), to give antibody-antigen complexes. The profile or pattern formed by the antigen-antibody complexes, termed an antibody fingerprint, when revealed by an effective amount of an appropriate detector molecule, is uniquely representative of a particular individual. The method can similarly by used to distinguish genetically, or otherwise similar individuals, or their body parts containing IS antibodies. Identification of inanimate objects, particularly security documents, is similarly affected by associating with the documents, an effective amount of a particular individual's IS antibodies, or conversely, a particular panel of antigens, and forming antibody-antigen complexes with a particular panel of antigens, or a particular individual's IS antibodies, respectively. One embodiment of the instant identification method, termed the blocked fingerprint assay, has applications in the area of allergy testing, autoimmune diagnostics and therapeutics, and the detection of environmental antigens such as pathogens, chemicals, and toxins.

  13. Recombinant Antibodies for Academia: A Practical Approach.

    PubMed

    Cosson, Pierre; Hartley, Oliver

    2016-12-21

    After several decades of optimization, phage display technology enables the routine isolation and production of recombinant monoclonal antibodies in vitro. As such it has the potential to provide the academic community with a vast, inexpensive and renewable supply of well-characterized reagents, reducing bottlenecks in basic science, helping increase reproducibility of experiments, and phasing out the use of animals for production and discovery of antibodies. Yet the overwhelming majority of fundamental research laboratories still use incompletely characterized antibodies developed in animals. In order to promote increased use of recombinant antibodies in academia, we have recently initiated an open source recombinant antibody facility in Geneva (http://www.unige.ch/antibodies). Here we describe our experience at the Geneva Antibody Facility: the various techniques involved in isolation and production of antibodies, the strategic choices that we have made, and what we hope will be a bright future for this project as part of a growing movement in the scientific community to replace all animal-derived antibodies with recombinant antibodies.

  14. Serum Antibody Biomarkers for ASD

    DTIC Science & Technology

    2015-10-01

    with higher levels of communication problems among the ASD boys. 2. Identify the antibody recognized by the ASD1 peptoid. We have pulled down the...56 kD on the gel, and greater amount pulled down from the ASD pooled serum vs. TD pooled serum. Future study will be required to identify the...between ASD, TD and AM sera. - A peptoid, ASD1, has been identified that binds significantly lower levels of IgG1 in ASD boys vs. TD boys. - Pull- down

  15. A simpler sort of antibody.

    PubMed Central

    Masat, L; Wabl, M; Johnson, J P

    1994-01-01

    The monoclonal antibody NEMO is directed against a molecule expressed by human cells of the melanocytic lineage. Although obtained by conventional immunization and fusion procedures, NEMO consists solely of kappa light chain. SDS/PAGE analysis indicates that the kappa chains are present as both monomers and dimers. When these two forms were separated by gel filtration, only the monomeric form bound antigen. As kappa light chains from the myeloma MOPC-41 and the hybridoma MORK do not bind to the melanocytic cells, we conclude that the binding specificity of NEMO resides in the variable region. Images PMID:8302862

  16. [Evolution of monoclonal antibodies in cancer treatment].

    PubMed

    Kubczak, Małgorzata; Rogalińska, Małgorzata

    2016-01-01

    Since late 90s of last century the new age of directed therapy began using mainly biological constructs produced in rodents called monoclonal antibodies. The side effects of monoclonal antibodies were a challenge for pharmaceutical companies to improve the biological properties of these biological drugs. The humanization of monoclonal constructs was an idea to improve monoclonal antibodies next generation activity cancer cell reduction in humans. Moreover for some other patients sensitive for monoclonal antibodies therapy could also potentially induce immunological differences that might imply on human health. The new idea related to monoclonal antibodies was to design a small molecule constructs of nanoantibodies with ability to enter into cells. Such small molecules could find their targets inside human cells, even in nuclei leading to differences in cancer cells expression. The existing knowledge on monoclonal antibodies as well as directed activity of nanoantibodies could improve anticancer treatment efficancy of diseases.

  17. Selection, design, and engineering of therapeutic antibodies.

    PubMed

    Presta, Leonard G

    2005-10-01

    mAbs account for an increasing portion of marketed human biological therapeutics. As a consequence, the importance of optimal selection, design, and engineering of these not only has expanded in the past 2 decades but also is now coming into play as a competitive factor. This review delineates the 4 basic areas for optimal therapeutic antibody selection and provides examples of the increasing number of considerations necessary for, and options available for, antibody design. Though some of the advances in antibody technology (eg, antibodies derived from phage-display libraries) have already made it to market, other more recent advances, such as engineering antibodies for enhanced effector functions, may not be far behind, especially given the increasing competition for therapeutic antibodies to the same target (eg, anti-CD20 and anti-TNF-alpha).

  18. Antibody Based Imaging Strategies of Cancer

    PubMed Central

    Warram, Jason M; de Boer, Esther; Sorace, Anna G; Chung, Thomas K; Kim, Hyunki; Pleijhuis, Rick G; van Dam, Gooitzen M; Rosenthal, Eben L

    2014-01-01

    Although mainly developed for preclinical research and therapeutic use, antibodies have high antigen specificity, which can be used as a courier to selectively deliver a diagnostic probe or therapeutic agent to cancer. It is generally accepted that the optimal antigen for imaging will depend on both the expression in the tumor relative to normal tissue and the homogeneity of expression throughout the tumor mass and between patients. For the purpose of diagnostic imaging, novel antibodies can be developed to target antigens for disease detection, or current FDA-approved antibodies can be repurposed with the covalent addition of an imaging probe. Reuse of therapeutic antibodies for diagnostic purposes reduces translational costs since the safety profile of the antibody is well defined and the agent is already available under conditions suitable for human use. In this review, we will explore a wide range of antibodies and imaging modalities that are being translated to the clinic for cancer identification and surgical treatment. PMID:24913898

  19. Phase Separation in Solutions of Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Benedek, George; Wang, Ying; Lomakin, Aleksey; Latypov, Ramil

    2012-02-01

    We report the observation of liquid-liquid phase separation (LLPS) in a solution of humanized monoclonal antibodies, IgG2, and the effects of human serum albumin, a major blood protein, on this phase separation. We find a significant reduction of phase separation temperature in the presence of albumin, and a preferential partitioning of the albumin into the antibody-rich phase. We provide a general thermodynamic analysis of the antibody-albumin mixture phase diagram and relate its features to the magnitude of the effective inter-protein interactions. Our analysis suggests that additives (HSA in this report), which have moderate attraction with antibody molecules, may be used to forestall undesirable protein condensation in antibody solutions. Our findings are relevant to understanding the stability of pharmaceutical solutions of antibodies and the mechanisms of cryoglobulinemia.

  20. Peptide Antibodies: Past, Present, and Future.

    PubMed

    Houen, Gunnar

    2015-01-01

    Peptide antibodies recognize epitopes with amino acid residues adjacent in sequence ("linear" epitopes). Such antibodies can be made to virtually any sequence and have been immensely important in all areas of molecular biology and diagnostics due to their versatility and to the rapid growth in protein sequence information. Today, peptide antibodies can be routinely and rapidly made to large numbers of peptides, including peptides with posttranslationally modified residues, and are used for immunoblotting, immunocytochemistry, immunohistochemistry, and immunoassays. In the future, peptide antibodies will continue to be immensely important for molecular biology, TCR- and MHC-like peptide antibodies may be produced routinely, peptide antibodies with predetermined conformational specificities may be designed, and peptide-based vaccines may become part of vaccination programs.

  1. Factors determining antibody distribution in tumors.

    PubMed

    Thurber, Greg M; Schmidt, Michael M; Wittrup, K Dane

    2008-02-01

    The development of antibody therapies for cancer is increasing rapidly, primarily owing to their specificity. Antibody distribution in tumors is often extremely uneven, however, leading to some malignant cells being exposed to saturating concentrations of antibody, whereas others are completely untargeted. This is detrimental because large regions of cells escape therapy, whereas other regions might be exposed to suboptimal concentrations that promote a selection of resistant mutants. The distribution of antibody depends on a variety of factors, including dose, affinity, antigens per cell and molecular size. Because these parameters are often known or easily estimated, a quick calculation based on simple modeling considerations can predict the uniformity of targeting within a tumor. Such analyses should enable experimental researchers to identify in a straightforward way the limitations in achieving evenly distributed antibody, and design and test improved antibody therapeutics more rationally.

  2. Antibody to dihydropyridine calcium entry blockers

    SciTech Connect

    Thayer, S.; Minaskanian, G.; Fairhurst, A.

    1986-03-05

    Antibodies that recognize dihydropyridine calcium entry blockers were elicited from rabbits. A sensitive and specific radioimmunoassay for dihydropyridines was developed and its specificity compared to the DHP binding site in skeletal muscle membranes. The antibody bound (/sup 3/H)nitrendipine with a higher affinity (K/sub D/ = 0.155 nM) than did the DHP receptor of skeletal muscle (K/sub D/ = 1-3 nM). However, in contrast to the DHP receptor, the antibody recognized only those DHP drugs with meta-nitrophenyl substituents at the 4-position on the DHP ring, and thus reflected the meta position of the nitro group on the DHP hapten used as an antigen. Both the antibody and the receptor exhibited stereospecificity, with each site recognizing the (+) isomer of nicardipine as the more potent. This antibody should prove useful in the studies of some potentially irreversible DHP molecules and for use in the production of anti-idotype antibodies.

  3. [Improved IgG Antibody Diagnostics of Feather Duvet Lung by an Antibody Screening Test].

    PubMed

    Sennekamp, J; Lehmann, E

    2015-11-01

    The underdiagnosed feather duvet lung, an extrinsic allergic alveolitis (hypersensitivity pneumonitis) caused by duck and goose feathers, can be more frequently diagnosed, if duck and goose feather antibodies are included in the panel of the routinely applied IgG antibody screening test. This does not necessarily require extending the screening test to include duck and goose feather antigens. By analysing 100 sera with duck and goose antibodies we found that the commonly used pigeon and budgerigar antibodies can also screen for feather duvet antibodies. All examined sera lacking pigeon and budgerigar antibodies also lacked clear-cut duck and goose feather antibodies. The examined sera with strong pigeon or budgerigar antibodies always also contained feather duvet antibodies. However, sera with medium or low concentrated pigeon or budgerigar antibodies are not always associated with feather duvet antibodies. In the light of these observations, we find that 71% of the duck and goose antibody analyses would be dispensable without essential loss of quality, if the results of screening for pigeon and budgerigar antibodies were incorporated into the procedure of a step-by- step diagnostics.

  4. Somatic diversification of antibody responses

    SciTech Connect

    Zheng, B.; Kelsoe, G.; Han, S.

    1996-01-01

    The humoral immune response is the culmination of a complex series of cellular interactions and migrations that define specific pathways of antigen-driven B cell differentiation. There are about 5 x 10{sup 8} and 10{sup 12} B lymphocyte lineage cells in the mouse and human, respectively, after the immune system has been established. B lymphocytes perceive antigen in their environment by virtue of their surface receptors (B cell receptor; BCR), in which membrane-associated immunoglobulin (mIg) is the antigen recognition substructure and the associated Ig{alpha} and Ig{beta} molecules transduce the activation signal. mIgs have extensive structural homology to immunoglobulins which are secreted by differentiated daughter cells following antigen stimulation. The specificity of a given BCR or antibody is created by a programmed successive process of gene rearrangements, first of D to J segments, then of V to DJ segments of the Ig heavy (H)-chain gene loci, and finally, of V to J segments of the Ig light (L)-chain gene loci. V,D, and J elements are assembled in a enormous number of combinations; variability is further achieved at the break-points of joining segments, including the insertion of non-germline-encoded nucleotide (N)sequences at the borders of V(D)J points, hence the almost unlimited specificities of the B cells or antibody repertoire. 129 refs.

  5. Monoclonal Antibodies for Lipid Management.

    PubMed

    Feinstein, Matthew J; Lloyd-Jones, Donald M

    2016-07-01

    In recent years, biochemical and genetic studies have identified proprotein convertase subtilisin/kexin type 9 (PCSK9) as a major mediator of low-density lipoprotein cholesterol (LDL-c) levels and thereby a potential novel target for reducing risk of coronary heart disease (CHD). These observations led to the development of PCSK9 inhibitors, which lower LDL-c levels more than any other non-invasive lipid-lowering therapy presently available. The PCSK9 inhibitors furthest along in clinical trials are subcutaneously injected monoclonal antibodies. These PCSK9 inhibitors have demonstrated LDL-c-lowering efficacy with acceptable safety in phase III clinical trials and may offer a useful therapy in addition to maximally tolerated HMG-CoA reductase inhibitors (statins) in certain patient groups. Longer-term data are required to ensure sustained efficacy and safety of this new class of medications. This review provides an overview of the biology, genetics, development, and clinical trials of monoclonal antibodies designed to inhibit PCSK9.

  6. Single-domain antibodies for biomedical applications.

    PubMed

    Krah, Simon; Schröter, Christian; Zielonka, Stefan; Empting, Martin; Valldorf, Bernhard; Kolmar, Harald

    2016-01-01

    Single-domain antibodies are the smallest antigen-binding units of antibodies, consisting either only of one variable domain or one engineered constant domain that solely facilitates target binding. This class of antibody derivatives comprises naturally occurring variable domains derived from camelids and sharks as well as engineered human variable or constant antibody domains of the heavy or light chain. Because of their high affinity and specificity as well as stability, small size and benefit of multiple re-formatting opportunities, those molecules emerged as promising candidates for biomedical applications and some of these entities have already proven to be successful in clinical development.

  7. STUDIES ON THE CONTROL OF ANTIBODY SYNTHESIS

    PubMed Central

    Brody, Neil I.; Walker, J. Geoffrey; Siskind, Gregory W.

    1967-01-01

    In the system studied, antigenic competition between two haptenic determinants was found to be of the same extent whether the haptens were on the same or on separate carrier molecules. Suppression of antibody formation to one determinant by administration of passive antibody partially eliminated the depressive effects of antigenic competition when the two haptens were located on separate carrier molecules but had no effect on antibody production to the second hapten when the two determinants were present on the same molecule. The results are discussed in terms of the mechanisms of suppression, antigenic competition, and control of antibody formation. PMID:4165502

  8. Preparation of astatine-labeled monoclonal antibodies

    SciTech Connect

    Milesz, S.; Norseev, Yu.V.; Szucs, Z. |

    1995-07-01

    In the cationic state astatine forms a stable complex with diethylenetriaminepentaacetic acid. Thanks to this complex, astatine can be bound to monoclonal antibodies of the RYa{sub 1} type. The most favorable conditions for preparing astatine-labeled antibodies are established. The chromatographic analysis and electromigration experiments showed that astatine is firmly linked to a biomolecule in vitro and it did not escape from labeled monoclonal antibodies even under treatment with such highly effective astatine-complexing agent as thiourea. The immune activity of astatine-labeled antibodies did not change even after 20 h.

  9. Monoclonal Antibody That Defines Human Myoepithelium

    NASA Astrophysics Data System (ADS)

    Dairkee, Shahnaz Hashmi; Blayney, Carlene; Smith, Helene S.; Hackett, Adeline J.

    1985-11-01

    We have isolated a mouse monoclonal antibody that, upon immunohistochemical localization in frozen sections, displays specificity for human myoepithelial cells in the resting mammary gland, sweat glands, and salivary glands. Furthermore, this antibody was strongly and homogeneously reactive with frozen sections of 3 of 60 breast carcinoma specimens. Using immunolocalization techniques in conjunction with polyacrylamide gel electrophoresis, we have determined that the reactivity of this monoclonal antibody is directed toward a 51,000-dalton keratin polypeptide. The potential uses of this antibody in the prognosis of human mammary carcinoma and in understanding the role of the myoepithelium in development and differentiation are discussed.

  10. 6th Annual European Antibody Congress 2010

    PubMed Central

    2011-01-01

    The 6th European Antibody Congress (EAC), organized by Terrapinn Ltd., was held in Geneva, Switzerland, which was also the location of the 4th and 5th EAC.1,2 As was the case in 2008 and 2009, the EAC was again the largest antibody congress held in Europe, drawing nearly 250 delegates in 2010. Numerous pharmaceutical and biopharmaceutical companies active in the field of therapeutic antibody development were represented, as were start-up and academic organizations and representatives from the US Food and Drug Administration (FDA). The global trends in antibody research and development were discussed, including success stories of recent marketing authorizations of golimumab (Simponi®) and canakinumab (Ilaris®) by Johnson & Johnson and Novartis, respectively, updates on antibodies in late clinical development (obinutuzumab/GA101, farletuzumab/MORAb-003 and itolizumab/T1 h, by Glycart/Roche, Morphotek and Biocon, respectively) and success rates for this fast-expanding class of therapeutics (Tufts Center for the Study of Drug Development). Case studies covering clinical progress of girentuximab (Wilex), evaluation of panobacumab (Kenta Biotech), characterization of therapeutic antibody candidates by protein microarrays (Protagen), antibody-drug conjugates (sanofi-aventis, ImmunoGen, Seattle Genetics, Wyeth/Pfizer), radio-immunoconjugates (Bayer Schering Pharma, Université de Nantes) and new scaffolds (Ablynx, AdAlta, Domantis/GlaxoSmithKline, Fresenius, Molecular Partners, Pieris, Scil Proteins, Pfizer, University of Zurich) were presented. Major antibody structural improvements were showcased, including the latest selection engineering of the best isotypes (Abbott, Pfizer, Pierre Fabre), hinge domain (Pierre Fabre), dual antibodies (Abbott), IgG-like bispecific antibodies (Biogen Idec), antibody epitope mapping case studies (Eli Lilly), insights in FcγRII receptor (University of Cambridge), as well as novel tools for antibody fragmentation (Genovis). Improvements

  11. Reducing the cost of HIV antibody testing.

    PubMed

    Tamashiro, H; Maskill, W; Emmanuel, J; Fauquex, A; Sato, P; Heymann, D

    1993-07-10

    Available tests to detect antibody to human immunodeficiency virus (HIV) have a range of applications, and injudicious selection and inappropriate use can add a significant financial burden to budgets for AIDS programmes in developing countries. There are several ways by which the cost of HIV antibody testing can be reduced; they include use of tests appropriate for existing laboratory capabilities; adoption of cost-effective testing strategies; pooling of serum samples before testing; and ensuring best possible purchase prices. Each approach can significantly reduce the cost of HIV antibody testing alone or in combination, which increases the potential sustainability of antibody testing programmes, even in settings of limited resources.

  12. Antibodies in myasthenia gravis and related disorders.

    PubMed

    Vincent, Angela; McConville, John; Farrugia, Maria Elena; Bowen, John; Plested, Paul; Tang, Teresa; Evoli, Amelia; Matthews, Ian; Sims, Gary; Dalton, Paolo; Jacobson, Leslie; Polizzi, Agata; Blaes, Frans; Lang, Bethan; Beeson, David; Willcox, Nick; Newsom-Davis, John; Hoch, Werner

    2003-09-01

    Acetylcholine receptor (AChR) antibodies are present in around 85% of patients with myasthenia gravis (MG) as measured by the conventional radioimmunoprecipitation assay. Antibodies that block the fetal form of the AChR are occasionally present in mothers who develop MG after pregnancy, especially in those whose babies are born with arthrogryposis multiplex congenita. The antibodies cross the placenta and block neuromuscular transmission, leading to joint deformities and often stillbirth. In these mothers, antibodies made in the thymus are mainly specific for fetal AChR and show restricted germline origins, suggesting a highly mutated clonal response; subsequent spread to involve adult AChR could explain development of maternal MG in those cases who first present after pregnancy. In the 15% of "seronegative" MG patients without AChR antibodies (SNMG), there are serum factors that increase AChR phosphorylation and reduce AChR function, probably acting via a different membrane receptor. These factors are not IgG and could be IgM or even non-Ig serum proteins. In a proportion of SNMG patients, however, IgG antibodies to the muscle-specific kinase, MuSK, are present. These antibodies are not found in AChR antibody-positive MG and are predominantly IgG4. MuSK antibody positivity appears to be associated with more severe bulbar disease that can be difficult to treat effectively.

  13. Monoclonal Antibodies against the Drosophila Nervous System

    NASA Astrophysics Data System (ADS)

    Fujita, Shinobu C.; Zipursky, Stephen L.; Benzer, Seymour; Ferrus, Alberto; Shotwell, Sandra L.

    1982-12-01

    A panel of 148 monoclonal antibodies directed against Drosophila neural antigens has been prepared by using mice immunized with homogenates of Drosophila tissue. Antibodies were screened immunohistochemically on cryostat sections of fly heads. A large diversity of staining patterns was observed. Some antigens were broadly distributed among tissues; others were highly specific to nerve fibers, neuropil, muscle, the tracheal system, cell nuclei, photoreceptors, or other structures. The antigens for many of the antibodies have been identified on immunoblots. Monoclonal antibodies that identify specific molecules within the nervous system should prove useful in the study of the molecular genetics of neural development.

  14. Allo-antibody identification: a software approach!

    PubMed

    Tiwari, Aseem Kumar; Dara, Ravi C; Arora, Dinesh; Aggarwal, Geet; Rawat, Ganesh; Mitra, Subhasis; Prashant, Pandey K; Raina, Vimarsh

    2014-10-01

    Unexpected allo-antibody identification is difficult serological test requiring in-depth knowledge of antibody behavior, identification rules, knowledge of zygosity of antigens and dosage phenomenon. Software which uses an algorithm based on characteristics of antibodies is now available to interpret specificity of allo-antibody. A study was undertaken to evaluate the effectiveness of one such software (Resolvigen) for antibody identification compared with manual antibody identification method. The study was a retrospective observational study where 238 allo-antibody results were re-evaluated using Resolvigen software (Ortho Clinical Diagnostics, Johnson and Johnson, Raritan, NJ, USA) and agreement between manual and software approaches was studied. Resolvigen software was also evaluated for usefulness, ease of use and predicted future usage by administering investigators a questionnaire with Likert scale. Agreement between the results of manual and automated methods ranged from 98.6% for single antibody to 65% for two antibodies (p = 0.000). Resolvigen software came out as very useful, easy to use, and with high predicted future usage. This study concludes that Resolvigen can either replace manual method or be used as adjuvant to routine manual method. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Optimizing antibody expression: The nuts and bolts.

    PubMed

    Ayyar, B Vijayalakshmi; Arora, Sushrut; Ravi, Shiva Shankar

    2017-03-01

    Antibodies are extensively utilized entities in biomedical research, and in the development of diagnostics and therapeutics. Many of these applications require high amounts of antibodies. However, meeting this ever-increasing demand of antibodies in the global market is one of the outstanding challenges. The need to maintain a balance between demand and supply of antibodies has led the researchers to discover better means and methods for optimizing their expression. These strategies aim to increase the volumetric productivity of the antibodies along with the reduction of associated manufacturing costs. Recent years have witnessed major advances in recombinant protein technology, owing to the introduction of novel cloning strategies, gene manipulation techniques, and an array of cell and vector engineering techniques, together with the progress in fermentation technologies. These innovations were also highly beneficial for antibody expression. Antibody expression depends upon the complex interplay of multiple factors that may require fine tuning at diverse levels to achieve maximum yields. However, each antibody is unique and requires individual consideration and customization for optimizing the associated expression parameters. This review provides a comprehensive overview of several state-of-the-art approaches, such as host selection, strain engineering, codon optimization, gene optimization, vector modification and process optimization that are deemed suitable for enhancing antibody expression. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Patterning of antibodies using flexographic printing.

    PubMed

    Phillips, Christopher O; Govindarajan, Sridhar; Hamblyn, Simon M; Conlan, R Steven; Gethin, David T; Claypole, Tim C

    2012-06-26

    Antibodies were patterned onto flexible plastic films using the flexographic printing process. An ink formulation was developed using high molecular weight polyvinyl alcohol in carbonate-bicarbonate buffer. In order to aid both antibody adhesion and the quality of definition in the printed features, a nitrocellulose coating was developed that was capable of being discretely patterned, thus increasing the signal-to-noise ratio of an antibody array. Printing antibody features such as dots, squares, text, and fine lines were reproduced effectively. Furthermore, this process could be easily adapted for printing of other biological materials, including, but not limited to, enzymes, DNA, proteins, aptamers, and cells.

  17. Monoclonal antibodies as diagnostics; an appraisal.

    PubMed

    Siddiqui, M Z

    2010-01-01

    Ever since the development of Hybridoma Technology in 1975 by Kohler and Milstein, our vision for antibodies as tools for research for prevention, detection and treatment of diseases, vaccine production, antigenic characterization of pathogens and in the study of genetic regulation of immune responses and disease susceptibility has been revolutionized. The monoclonal antibodies being directed against single epitopes are homogeneous, highly specific and can be produced in unlimited quantities. In animal disease diagnosis, they are very useful for identification and antigenic characterization of pathogens. Monoclonal antibodies have tremendous applications in the field of diagnostics, therapeutics and targeted drug delivery systems, not only for infectious diseases caused by bacteria, viruses and protozoa but also for cancer, metabolic and hormonal disorders. They are also used in the diagnosis of lymphoid and myeloid malignancies, tissue typing, enzyme linked immunosorbent assay, radio immunoassay, serotyping of microorganisms, immunological intervention with passive antibody, antiidiotype inhibition, or magic bullet therapy with cytotoxic agents coupled with anti mouse specific antibody. Recombinant deoxyribonucleic acid technology through genetic engineering has successfully led to the possibility of reconstruction of monoclonal antibodies viz. chimeric antibodies, humanized antibodies and complementarily determining region grafted antibodies and their enormous therapeutic use.

  18. Bioconjugation of Antibodies to Horseradish Peroxidase (HRP).

    PubMed

    Hnasko, Robert M

    2015-01-01

    The bioconjugation of an antibody to an enzymatic reporter such as horseradish peroxidase (HRP) affords an effective mechanism by which immunoassay detection of a target antigen can be achieved. The use of heterobifunctional cross-linkers to covalently link antibodies to HRP provides a simple and convenient means to maintain antibody affinity while imparting a functional reporter used for antigen detection. In this chapter, we describe a process by which Sulfo-SMCC is used to generate a stable maleimide-activated HRP that is reactive with sulfhydryl groups generated in antibodies by SATA-mediated thiolation.

  19. A general approach to antibody thermostabilization

    PubMed Central

    McConnell, Audrey D; Zhang, Xue; Macomber, John L; Chau, Betty; Sheffer, Joseph C; Rahmanian, Sorena; Hare, Eric; Spasojevic, Vladimir; Horlick, Robert A; King, David J; Bowers, Peter M

    2014-01-01

    Antibody engineering to enhance thermostability may enable further application and ease of use of antibodies across a number of different areas. A modified human IgG framework has been developed through a combination of engineering approaches, which can be used to stabilize antibodies of diverse specificity. This is achieved through a combination of complementarity-determining region (CDR)-grafting onto the stable framework, mammalian cell display and in vitro somatic hypermutation (SHM). This approach allows both stabilization and maturation to affinities beyond those of the original antibody, as shown by the stabilization of an anti-HA33 antibody by approximately 10°C and affinity maturation of approximately 300-fold over the original antibody. Specificities of 10 antibodies of diverse origin were successfully transferred to the stable framework through CDR-grafting, with 8 of these successfully stabilized, including the therapeutic antibodies adalimumab, stabilized by 9.9°C, denosumab, stabilized by 7°C, cetuximab stabilized by 6.9°C and to a lesser extent trastuzumab stabilized by 0.8°C. This data suggests that this approach may be broadly useful for improving the biophysical characteristics of antibodies across a number of applications. PMID:25517312

  20. Reconciling the structural attributes of avian antibodies.

    PubMed

    Conroy, Paul J; Law, Ruby H P; Gilgunn, Sarah; Hearty, Stephen; Caradoc-Davies, Tom T; Lloyd, Gordon; O'Kennedy, Richard J; Whisstock, James C

    2014-05-30

    Antibodies are high value therapeutic, diagnostic, biotechnological, and research tools. Combinatorial approaches to antibody discovery have facilitated access to unique antibodies by surpassing the diversity limitations of the natural repertoire, exploitation of immune repertoires from multiple species, and tailoring selections to isolate antibodies with desirable biophysical attributes. The V-gene repertoire of the chicken does not utilize highly diverse sequence and structures, which is in stark contrast to the mechanism employed by humans, mice, and primates. Recent exploitation of the avian immune system has generated high quality, high affinity antibodies to a wide range of antigens for a number of therapeutic, diagnostic and biotechnological applications. Furthermore, extensive examination of the amino acid characteristics of the chicken repertoire has provided significant insight into mechanisms employed by the avian immune system. A paucity of avian antibody crystal structures has limited our understanding of the structural consequences of these uniquely chicken features. This paper presents the crystal structure of two chicken single chain fragment variable (scFv) antibodies generated from large libraries by phage display against important human antigen targets, which capture two unique CDRL1 canonical classes in the presence and absence of a non-canonical disulfide constrained CDRH3. These structures cast light on the unique structural features of chicken antibodies and contribute further to our collective understanding of the unique mechanisms of diversity and biochemical attributes that render the chicken repertoire of particular value for antibody generation.

  1. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V

    2013-08-06

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides a method of inhibiting the growth of tumor cells comprising contacting said tumor cells with an appropriate amount of monoclonal antibody 8H9 or a derivative thereof.

  2. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V.

    2010-06-15

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides a method of inhibiting the growth of tumor cells comprising contacting said tumor cells with an appropriate amount of monoclonal antibody 8H9 or a derivative thereof.

  3. Antibody engineering and therapeutics, The Annual Meeting of the Antibody Society: December 8-12, 2013, Huntington Beach, CA.

    PubMed

    Almagro, Juan Carlos; Gilliland, Gary L; Breden, Felix; Scott, Jamie K; Sok, Devin; Pauthner, Matthias; Reichert, Janice M; Helguera, Gustavo; Andrabi, Raiees; Mabry, Robert; Bléry, Mathieu; Voss, James E; Laurén, Juha; Abuqayyas, Lubna; Barghorn, Stefan; Ben-Jacob, Eshel; Crowe, James E; Huston, James S; Johnston, Stephen Albert; Krauland, Eric; Lund-Johansen, Fridtjof; Marasco, Wayne A; Parren, Paul W H I; Xu, Kai Y

    2014-01-01

    The 24th Antibody Engineering & Therapeutics meeting brought together a broad range of participants who were updated on the latest advances in antibody research and development. Organized by IBC Life Sciences, the gathering is the annual meeting of The Antibody Society, which serves as the scientific sponsor. Preconference workshops on 3D modeling and delineation of clonal lineages were featured, and the conference included sessions on a wide variety of topics relevant to researchers, including systems biology; antibody deep sequencing and repertoires; the effects of antibody gene variation and usage on antibody response; directed evolution; knowledge-based design; antibodies in a complex environment; polyreactive antibodies and polyspecificity; the interface between antibody therapy and cellular immunity in cancer; antibodies in cardiometabolic medicine; antibody pharmacokinetics, distribution and off-target toxicity; optimizing antibody formats for immunotherapy; polyclonals, oligoclonals and bispecifics; antibody discovery platforms; and antibody-drug conjugates.

  4. Immune Antibody Libraries: Manipulating The Diverse Immune Repertoire for Antibody Discovery.

    PubMed

    Lim, Theam Soon; Chan, Soo Khim

    2016-01-01

    Antibody phage display is highly dependent on the availability of antibody libraries. There are several forms of libraries depending mainly on the origin of the source materials. There are three major classes of libraries, mainly the naïve, immune and synthetic libraries. Immune antibody libraries are designed to isolate specific and high affinity antibodies against disease antigens. The pre-exposure of the host to an infection results in the production of a skewed population of antibodies against the particular infection. This characteristic takes advantage of the in vivo editing machinery to generate bias and specific immune repertoire. The skewed but diverse repertoire of immune libraries has been adapted successfully in the generation of antibodies against a wide range of diseases. We envisage immune antibody libraries to play a greater role in the discovery of antibodies for diseases in the near future. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  5. Lack of in Vivo Antibody Dependent Cellular Cytotoxicity with Antibody Containing Gold Nanoparticles

    PubMed Central

    2016-01-01

    Antibody-dependent cellular cytotoxicity (ADCC) is a cytolytic mechanism that can elicit in vivo antitumor effects and can play a significant role in the efficacy of antibody treatments for cancer. Here, we prepared cetuximab, panitumumab, and rituximab containing gold nanoparticles and investigated their ability to produce an ADCC effect in vivo. Cetuximab treatment of EGFR-expressing H1975 tumor xenografts showed significant tumor regression due to the ADCC activity of the antibody in vivo, while the control antibody, panitumumab, did not. However, all three antibody containing nanoparticles are not able to suppress tumor growth in the same in vivo mouse model. The antibody containing nanoparticles localized in the tumors and did not suppress the immune function of the animals, so the lack of tumor growth suppression of the cetuximab containing nanoparticle suggests that immobilizing antibodies onto a nanoparticle significantly decreases the ability of the antibody to promote an ADCC response. PMID:25879583

  6. Monoclonal antibodies with group specificity toward sulfonamides: Selection of hapten and antibody selectivity

    USDA-ARS?s Scientific Manuscript database

    Although many antibodies to sulfonamides have been generated, immunoassays based on the current available antibodies for large multi-sulfonamide screening programs have properties dependent on the immunizing hapten structure and have always suffered from high selectivity for individual sulfonamides....

  7. Not All Antibodies Are Created Equal: Factors That Influence Antibody Mediated Rejection

    PubMed Central

    Butler, Carrie L.; Valenzuela, Nicole M.; Thomas, Kimberly A.

    2017-01-01

    Consistent with Dr. Paul Terasaki's “humoral theory of rejection” numerous studies have shown that HLA antibodies can cause acute and chronic antibody mediated rejection (AMR) and decreased graft survival. New evidence also supports a role for antibodies to non-HLA antigens in AMR and allograft injury. Despite the remarkable efforts by leaders in the field who pioneered single antigen bead technology for detection of donor specific antibodies, a considerable amount of work is still needed to better define the antibody attributes that are associated with AMR pathology. This review highlights what is currently known about the clinical context of pre and posttransplant antibodies, antibody characteristics that influence AMR, and the paths after donor specific antibody production (no rejection, subclinical rejection, and clinical dysfunction with AMR). PMID:28373996

  8. Similar Idiotypes in Antibody-Forming Cells and in Cells Synthesizing Immunoglobulins Without Detectable Antibody Function

    PubMed Central

    Cazenave, P. -A.; Ternynck, T.; Avrameas, S.

    1974-01-01

    The occurrence of immunoglobulins with and without antibody specificity and with and without idiotypic specificity was studied, by use of enzyme-labeled antigen and antibodies, in lymph node cells of rabbits immunized with horse-radish peroxidase and hen ovalbumin. Some cells, containing immunoglobulins without detectable antibody function, were shown to contain idiotypes similar to those found in antibody-producing cells. PMID:4140504

  9. Antibody i-Patch prediction of the antibody binding site improves rigid local antibody-antigen docking.

    PubMed

    Krawczyk, Konrad; Baker, Terry; Shi, Jiye; Deane, Charlotte M

    2013-10-01

    Antibodies are a class of proteins indispensable for the vertebrate immune system. The general architecture of all antibodies is very similar, but they contain a hypervariable region which allows millions of antibody variants to exist, each of which can bind to different molecules. This binding malleability means that antibodies are an increasingly important category of biopharmaceuticals and biomarkers. We present Antibody i-Patch, a method that annotates the most likely antibody residues to be in contact with the antigen. We show that our predictions correlate with energetic importance and thus we argue that they may be useful in guiding mutations in the artificial affinity maturation process. Using our predictions as constraints for a rigid-body docking algorithm, we are able to obtain high-quality results in minutes. Our annotation method and re-scoring system for docking achieve their predictive power by using antibody-specific statistics. Antibody i-Patch is available from http://www.stats.ox.ac.uk/research/proteins/resources.

  10. Oral immunization with xenogeneic antibodies stimulates the production of systemic and mucosal anti-idiotypic antibodies.

    PubMed Central

    Collins, A M; Roberton, D M; Hosking, C S; Flannery, G R

    1991-01-01

    The humoral and mucosal immune responses to oral immunization with xenogeneic antibodies were studied using an animal model in which female rabbits were fed daily doses of the MOPC-315 murine IgA antibody, and were mated during the course of the feeding programme. Serum and colostrum samples were assayed for the presence of anti-idiotypic antibodies by ELISA assay, before and after depletion of anti-IgA antibodies, by affinity chromatography using another murine IgA idiotype. It was shown that all animals responded to exposure to the MOPC-315 idiotype with the production of serum anti-murine immunoglobulin antibodies and that four of six animals produced serum anti-idiotypic antibodies. That the immune response included antibodies directed against the antigen-binding site was confirmed by competition ELISA assay. Mucosal IgG and IgA anti-immunoglobulin antibodies were present in milk from all antibody-fed rabbits tested, and IgA anti-idiotypic antibodies were detectable in the colostrum of one rabbit. The results provide some support for the hypothesis that human exposure to xenogeneic antibodies, most commonly bovine milk immunoglobulins, may provoke the production of anti-idiotypic antibodies, and that such exposure may lead to disturbances of immune regulation. PMID:1916890

  11. Passive antibody transfer in chickens to model maternal antibody after avian influenza vaccination

    USDA-ARS?s Scientific Manuscript database

    Birds transfer maternal antibodies (MAb) to their offspring through the egg yolk where the antibody is absorbed and enters the circulatory system. These maternal antibodies, depending on the pathogen, can provide early protection from some diseases, but it may also interfere with the vaccination re...

  12. Passive antibody transfer in chickens to model maternal antibody after avian influenza vaccination

    USDA-ARS?s Scientific Manuscript database

    Birds transfer maternal antibodies (MAb) to their offspring through the egg yolk where the antibody is absorbed and enters the circulatory system. Maternal antibodies provide early protection from disease, but may interfere with the vaccination efficacy in the chick. MAb are thought to interfere wit...

  13. Antibodies to watch in 2017

    PubMed Central

    Reichert, Janice M.

    2017-01-01

    ABSTRACT Over 50 investigational monoclonal antibody (mAb) therapeutics are currently undergoing evaluation in late-stage clinical studies, which is expected to drive a trend toward first marketing approvals of at least 6–9 mAbs per year in the near-term. In the United States (US), a total of 6 and 9 mAbs were granted first approvals during 2014 and 2015, respectively; all these products are also approved in the European Union (EU). As of December 1, 2016, 6 mAbs (atezolizumab, olaratumab, reslizumab, ixekizumab, bezlotoxumab, oblitoxaximab) had been granted first approvals during 2016 in either the EU or US. Brodalumab, was granted a first approval in Japan in July 2016. Regulatory actions on marketing applications for brodalumab in the EU and US are not expected until 2017. In 2017, first EU or US approvals may also be granted for at least nine mAbs (ocrelizumab, avelumab, Xilonix, inotuzumab ozogamicin, dupilumab, sirukumab, sarilumab, guselkumab, romosozumab) that are not yet approved in any country. Based on announcements of company plans for regulatory submissions and the estimated completion dates for late-stage clinical studies, and assuming the study results are positive, marketing applications for at least 6 antibody therapeutics (benralizumab, tildrakizumab, emicizumab, galcanezumab, ibalizumab, PRO-140) that are now being evaluated in late-stage clinical studies may be submitted during December 2016* or 2017. Other ‘antibodies to watch' in 2017 include 20 mAbs are undergoing evaluation in pivotal studies that have estimated primary completion dates in late 2016 or during 2017. Of these, 5 mAbs are for cancer (durvalumab, JNJ-56022473, ublituximab, anetumab ravtansine, glembatumumab vedotin) and 15 mAbs are for non-cancer indications (caplacizumab, lanadelumab, roledumab, tralokinumab, risankizumab, SA237, emapalumab, suptavumab, erenumab, eptinezumab, fremanezumab, fasinumab, tanezumab, lampalizumab, brolucizumab). Positive results from these

  14. Antibodies to watch in 2017.

    PubMed

    Reichert, Janice M

    Over 50 investigational monoclonal antibody (mAb) therapeutics are currently undergoing evaluation in late-stage clinical studies, which is expected to drive a trend toward first marketing approvals of at least 6-9 mAbs per year in the near-term. In the United States (US), a total of 6 and 9 mAbs were granted first approvals during 2014 and 2015, respectively; all these products are also approved in the European Union (EU). As of December 1, 2016, 6 mAbs (atezolizumab, olaratumab, reslizumab, ixekizumab, bezlotoxumab, oblitoxaximab) had been granted first approvals during 2016 in either the EU or US. Brodalumab, was granted a first approval in Japan in July 2016. Regulatory actions on marketing applications for brodalumab in the EU and US are not expected until 2017. In 2017, first EU or US approvals may also be granted for at least nine mAbs (ocrelizumab, avelumab, Xilonix, inotuzumab ozogamicin, dupilumab, sirukumab, sarilumab, guselkumab, romosozumab) that are not yet approved in any country. Based on announcements of company plans for regulatory submissions and the estimated completion dates for late-stage clinical studies, and assuming the study results are positive, marketing applications for at least 6 antibody therapeutics (benralizumab, tildrakizumab, emicizumab, galcanezumab, ibalizumab, PRO-140) that are now being evaluated in late-stage clinical studies may be submitted during December 2016* or 2017. Other 'antibodies to watch' in 2017 include 20 mAbs are undergoing evaluation in pivotal studies that have estimated primary completion dates in late 2016 or during 2017. Of these, 5 mAbs are for cancer (durvalumab, JNJ-56022473, ublituximab, anetumab ravtansine, glembatumumab vedotin) and 15 mAbs are for non-cancer indications (caplacizumab, lanadelumab, roledumab, tralokinumab, risankizumab, SA237, emapalumab, suptavumab, erenumab, eptinezumab, fremanezumab, fasinumab, tanezumab, lampalizumab, brolucizumab). Positive results from these studies may

  15. Antibody humanization methods for development of therapeutic applications.

    PubMed

    Ahmadzadeh, Vahideh; Farajnia, Safar; Feizi, Mohammad Ali Hosseinpour; Nejad, Ramezan Ali Khavari

    2014-04-01

    Recombinant antibody technologies are rapidly becoming available and showing considerable clinical success. However, the immunogenicity of murine-derived monoclonal antibodies is restrictive in cancer immunotherapy. Humanized antibodies can overcome these problems and are considered to be a promising alternative therapeutic agent. There are several approaches for antibody humanization. In this article we review various methods used in the antibody humanization process.

  16. Antibody Characterization Process | Office of Cancer Clinical Proteomics Research

    Cancer.gov

    The goal of the NCI's Antibody Characterization Program (ACP) is to have three monoclonal antibodies produced for each successfully expressed/purified recombinant antigen and one antibody per peptide (1 to 3 peptides per protein). To date, over 4000 clones have been screened before selecting the current 393 antibodies. They are winnowed down based on the projected end use of the antibody.

  17. Testing for antineutrophil cytoplasmic antibodies.

    PubMed

    Savige, J

    2001-09-01

    The most common reason to request a test for antineutrophil cytoplasmic antibodies (ANCA) is to diagnose Wegener's granulomatosis and microscopic polyangiitis and to monitor inflammatory activity in these diseases. Several retrospective and prospective studies have suggested that the demonstration of ANCA lacks sensitivity and specificity, but these series have detected ANCA with neutrophil-indirect immunofluorescence alone, have used a disease classification that did not describe microscopic polyangiitis and have included patients with inactive disease. The 'International Consensus Statement on Testing and Reporting ANCA' has been developed to optimize the clinical relevance of ANCA testing by the adoption of standardized testing and reporting procedures. International collaborative efforts continue to focus on improving the tests for ANCA.

  18. Quality Issues of Research Antibodies

    PubMed Central

    Weller, Michael G.

    2016-01-01

    According to several recent studies, an unexpectedly high number of landmark papers seem to be not reproducible by independent laboratories. Nontherapeutic antibodies used for research, diagnostic, food analytical, environmental, and other purposes play a significant role in this matter. Although some papers have been published offering suggestions to improve the situation, they do not seem to be comprehensive enough to cover the full complexity of this issue. In addition, no obvious improvements could be noticed in the field as yet. This article tries to consolidate the remarkable variety of conclusions and suggested activities into a more coherent conception. It is concluded that funding agencies and journal publishers need to take first and immediate measures to resolve these problems and lead the way to a more sustainable way of bioanalytical research, on which all can rely with confidence. PMID:27013861

  19. Clinical considerations for biosimilar antibodies

    PubMed Central

    Mellstedt, Håkan

    2013-01-01

    Biosimilar agents are approximate copies of branded biologic therapies. Since the first biosimilar was authorized in the European Union in 2006, fifteen additional agents have been approved by the European Medicines Agency, including two biosimilar monoclonal antibodies (mAbs). Biosimilar mAbs represent a distinct class given their large molecular size, complex protein structure, and post-translational modifications. While guidelines have been established for the development, approval, and use of biosimilars, further scrutiny and discussion is necessary to fully understand their potential impact on clinical outcomes. This review takes a critical look at the structural complexity of biosimilar mABs, the feasibility of indication extrapolation, the impact of product variability on immunogenicity, the importance of comprehensive pharmacovigilance, and the potential for ongoing pharmacoeconomic impact. PMID:26217160

  20. The nature of antiphospholipid antibodies.

    PubMed

    Rauch, J; Janoff, A S

    1992-11-01

    Despite the striking clinical manifestations associated with antiphospholipid antibodies (aPL) the role of these autoantibodies in disease and the nature of their true "inducing" and "target" antigens remain elusive. To address these issues, we investigated the immunogenic potential of phospholipid structures. To date, phospholipid immunogens have included hexagonal (II) forms of phosphatidylethanolamine and mixtures of apolipoprotein H (beta 2-glycoprotein I) with cardiolipin. Both hexagonal (II) phosphatidylethanolamine and the cardiolipin/apolipoprotein H mixture were capable of inducing aPL with lupus anticoagulant activity. Bilayer phosphatidylethanolamine and cardiolipin in the absence of apolipoprotein H were nonimmunogenic. Our data support our views that specific phospholipid structures are recognized by the immune system and that such structures serve as inducing and/or target antigens in the pathogenesis of aPL in vivo.

  1. Antinuclear Antibody Negative Systemic Sclerosis

    PubMed Central

    Salazar, GA; Assassi, S; Wigley, F; Hummers, L; Varga, J; Hinchcliff, M; Khanna, D; Schiopu, E; Phillips, K; Furst, DE; Steen, V; Baron, M; Hudson, M; Taillefer, SS; Pope, J; Jones, N; Docherty, P; Khalidi, NA; Robinson, D; Simms, R; Silver, R; Frech, TM; Fessler, B; Molitor, J; Fritzler, M; Segal, B; Al-Kassab, F; Perry, M; Yang, J; Zamanian, S; Reveille, JD; Arnett, FC; Pedroza, C; Mayes, MD

    2014-01-01

    Objective To examine the demographic and clinical characteristics of systemic sclerosis (SSc) patients without antinuclear antibodies (ANA) compared to ANA positive patients. Methods SSc patients enrolled in the Scleroderma Family Registry and DNA Repository were included. Relevant demographic and clinical data were entered by participating sites or obtained by chart review. ANA and SSc related antibodies were determined in all investigated patients using commercially available kits at our laboratories. Results This study included 3249 patients, of whom 208 (6.4%) were ANA negative. The proportion of male patients was higher in the ANA negative group (OR 1.65; p=0.008). ANA negative patients experienced less vasculopathic manifestations of SSc. The percent predicted diffusing capacity of carbon monoxide (DLco) was higher in ANA negative patients (p=0.03). Pulmonary arterial hypertension (PAH) per right heart catheterization was less common in the ANA negative group (OR= 0.28; p=0.03). Furthermore, patients with negative ANA had a lower prevalence of telangiectasias and digital ulcers/pits (OR= 0.59; p=0.03 and OR=0.38; p=0.01, respectively). Although diffuse cutaneous involvement was more common, the modified Rodnan Skin Score (mRSS) was lower in the ANA negative group (2.4 points lower, p=0.05). Furthermore, they experienced more malabsorption (p=0.05). There was no difference in the frequency of pulmonary fibrosis or scleroderma renal crisis. All-cause mortality was not different between the two groups (p=0.28). Conclusions In conclusion, the results of this study suggest that SSc patients who are ANA negative constitute a distinct subset of SSc with less vasculopathy (less PAH, digital ulcers and fewer telangiectasias), a greater proportion of males and possibly, more frequent lower gastrointestinal involvement. PMID:25578738

  2. Microscale purification of antigen-specific antibodies

    PubMed Central

    Brown, Eric P.; Normandin, Erica; Osei-Owusu, Nana Yaw; Mahan, Alison E.; Chan, Ying N.; Lai, Jennifer I.; Vaccari, Monica; Rao, Mangala; Franchini, Genoveffa; Alter, Galit; Ackerman, Margaret E.

    2015-01-01

    Glycosylation of the Fc domain is an important driver of antibody effector function. While assessment of antibody glycoform compositions observed across total plasma IgG has identified differences associated with a variety of clinical conditions, in many cases it is the glycosylation state of only antibodies against a specific antigen or set of antigens that may be of interest, for example, in defining the potential effector function of antibodies produced during disease or after vaccination. Historically, glycoprofiling such antigen-specific antibodies in clinical samples has been challenging due to their low prevalence, the high sample requirement for most methods of glycan determination, and the lack of high-throughput purification methods. New methods of glycoprofiling with lower sample requirements and higher throughput have motivated the development of microscale and automatable methods for purification of antigen-specific antibodies from polyclonal sources such as clinical serum samples. In this work, we present a robot-compatible 96-well plate-based method for purification of antigen-specific antibodies, suitable for such population level glycosylation screening. We demonstrate the utility of this method across multiple antibody sources, using both purified plasma IgG and plasma, and across multiple different antigen types, with enrichment factors greater than 1000-fold observed. Using an on-column IdeS protease treatment, we further describe staged release of Fc and Fab domains, allowing for glycoprofiling of each domain. PMID:26078040

  3. Antibody-drug conjugates: Intellectual property considerations

    PubMed Central

    Storz, Ulrich

    2015-01-01

    Antibody-drug conjugates are highly complex entities that combine an antibody, a linker and a toxin. This complexity makes them demanding both technically and from a regulatory point of view, and difficult to deal with in their patent aspects. This article discusses different issues of patent protection and freedom to operate with regard to this promising new class of drugs. PMID:26292154

  4. Bioconjugation of antibodies to horseradish peroxidase (hrp)

    USDA-ARS?s Scientific Manuscript database

    The bioconjugation of an antibody to an enzymatic reporter such as horseradish peroxidase (HRP) affords an effective mechanism by which immunoassay detection of a target antigen can be achieved. The use of heterobifunctional cross—linkers to covalently link antibodies to HRP provides a simple and c...

  5. Anti-influenza M2e antibody

    DOEpatents

    Bradbury, Andrew M [Santa Fe, NM

    2011-12-20

    Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.

  6. Epstein-Barr Virus Antibodies Test

    MedlinePlus

    ... and services. Advertising & Sponsorship: Policy | Opportunities Epstein-Barr Virus (EBV) Antibody Tests Share this page: Was this ... EA-D IgG Ab Formal name: Epstein-Barr Virus Antibody to Viral Capsid Antigen, IgM, IgG; Epstein- ...

  7. Anti-influenza M2e antibody

    DOEpatents

    Bradbury, Andrew M.

    2013-04-16

    Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.

  8. Antibody-drug conjugates: Intellectual property considerations.

    PubMed

    Storz, Ulrich

    2015-01-01

    Antibody-drug conjugates are highly complex entities that combine an antibody, a linker and a toxin. This complexity makes them demanding both technically and from a regulatory point of view, and difficult to deal with in their patent aspects. This article discusses different issues of patent protection and freedom to operate with regard to this promising new class of drugs.

  9. Immunoglobulin classes of Aleutian disease virus antibody.

    PubMed Central

    Porter, D D; Porter, H G; Suffin, S C; Larsen, A E

    1984-01-01

    Aleutian disease virus (ADV) persistently infects mink and causes marked hypergammaglobulinemia. Immunoglobulin class-specific antisera were used to define the total immunoglobulin of each class by radial immunodiffusion and the immunoglobulin class of ADV-specific antibody by immunofluorescence in experimentally and naturally infected mink. Electrophoretic gamma globulin closely reflects the immunoglobulin G (IgG) level in mink, and the majority of the increased immunoglobulin and ADV antibody in infected mink is IgG. IgM becomes elevated within 6 days after infection, reaches peak levels by 15 to 18 days, and returns to normal by 60 days after infection. The first ADV antibody demonstrable is IgM, and most mink have virus-specific IgM antibody for at least 85 days postinfection. Serum IgA levels in normal mink are not normally distributed, and ADV infection causes a marked elevation of IgA. Low levels of ADV-specific IgA antibody can be shown throughout the course of infection. Failure of large amounts of virus-specific IgG antibody to inhibit the reaction of virus-specific IgM and IgA antibodies suggests that the various classes of antibodies are directed against spatially different antigenic determinants. The IgM and IgA were shown not to be rheumatoid factors. PMID:6319283

  10. How to successfully patent therapeutic antibodies.

    PubMed

    Lahrtz, Fritz

    2015-04-01

    Therapeutic antibodies have become an established class of drugs for the treatment of a variety of diseases, especially cancer and autoimmune/inflammatory disorders, and a sufficient patent protection is a prerequisite for their successful commercialization. As monoclonal antibodies and their therapeutic potential have been well known for decades, the mere production of yet another therapeutic antibody is in many jurisdictions not considered a patentable invention. In contrast, antibodies with novel structural features and/or improved properties may be patentable. When drafting the claims, care should be taken to obtain a broad patent scope that protects both the antibody of interest and related antibodies having the same functional features, thereby preventing competitors from marketing a functionally equivalent antibody. Furthermore, the application should contain experimental evidence showing the improved properties of the claimed antibody. After the filing of a priority patent application, patent protection should be initiated at least in countries that are of particular commercial importance. Subsequent inventions relating to novel uses, formulations, dosage regimens, and combinations with other treatment modalities should be protected by further patent applications to extend patent term. © 2015 Society for Laboratory Automation and Screening.

  11. Antibody humanization methods - a review and update.

    PubMed

    Safdari, Yaghoub; Farajnia, Safar; Asgharzadeh, Mohammad; Khalili, Masoumeh

    2013-01-01

    This article reviews recent advances achieved during recent years on various aspects of antibody humanization theories and techniques. Common methods for producing humanized antibodies including framework-homology-based humanization, germline humanization, complementary determining regions (CDR)-homology-based humanization and specificity determining residues (SDR) grafting, as well as advantages and disadvantages of each of these methods and their applications are discussed.

  12. Production of monoclonal antibodies to plant pathogens.

    PubMed

    Thornton, Christopher R

    2009-01-01

    The use of monoclonal antibodies in plant pathology has improved the quality and specificity of detection methods for diseases. Hybridoma technology allows the limitless production of highly specific antibodies which can be used to identify pathogens to the species or even sub-species level.

  13. 7th Annual European Antibody Congress 2011

    PubMed Central

    2012-01-01

    The 7th European Antibody Congress (EAC), organized by Terrapin Ltd., was again held in Geneva, Switzerland, following on the tradition established with the 4th EAC. The 2011 version of the EAC was attended by nearly 250 delegates who learned of the latest advances and trends in the global development of antibody-based therapeutics. The first day focused on advances in understanding structure-function relationships, choosing the best format, glycoengineering biobetter antibodies, improving the efficacy and drugability of mAbs and epitope mapping. On the second day, the discovery of novel targets for mAb therapy, clinical pipeline updates, use of antibody combinations to address resistance, generation and identification of mAbs against new targets and biosimilar mAb development were discussed. Antibody-drug conjugates, domain antibodies and new scaffolds and bispecific antibodies were the topics of the third day. In total, nearly 50 speakers provided updates of programs related to antibody research and development on-going in the academic, government and commercial sectors. PMID:22453093

  14. Transplacental Chikungunya Virus Antibody Kinetics, Thailand

    PubMed Central

    Endy, Timothy P.; Simasathien, Sriluck; Kerdpanich, Angkool; Polprasert, Napuschon; Aree, Chanchai; Vaughn, David W.; Nisalak, Ananda

    2006-01-01

    Antibodies to chikungunya virus were detected by hemagglutination-inhibition assay in 33.6% of 2,000 infants' cord sera at delivery. Follow-up of 24 seropositive infants showed that the half-life of antibody persistence was 35.5 days. Chikungunya virus infection is common in Thailand, and routine use of diagnostic assays is needed. PMID:17283634

  15. Antibody Gene Transfer for HIV Immunoprophylaxis

    PubMed Central

    Balazs, Alejandro B.; West, Anthony P.

    2015-01-01

    Antibody gene transfer, which involves the delivery of genes that encode potent, broadly neutralizing anti-HIV antibodies, is a promising new strategy to prevent HIV infection. A satellite symposium at the AIDS Vaccine 2012 conference brought together many of the groups working in this field. PMID:23238748

  16. Monoclonal Antibody Therapy for Advanced Neuroblastoma

    Cancer.gov

    NCI is sponsoring two clinical trials of a monoclonal antibody called ch14.18, in combination with other drugs, to see if the antibody may be helpful for children or young adults (up to age 21) with relapsed or refractory neuroblastoma.

  17. Methods for Selecting Phage Display Antibody Libraries.

    PubMed

    Jara-Acevedo, Ricardo; Diez, Paula; Gonzalez-Gonzalez, Maria; Degano, Rosa Maria; Ibarrola, Nieves; Gongora, Rafael; Orfao, Alberto; Fuentes, Manuel

    2016-01-01

    The selection process aims sequential enrichment of phage antibody display library in clones that recognize the target of interest or antigen as the library undergoes successive rounds of selection. In this review, selection methods most commonly used for phage display antibody libraries have been comprehensively described.

  18. Microscale purification of antigen-specific antibodies.

    PubMed

    Brown, Eric P; Normandin, Erica; Osei-Owusu, Nana Yaw; Mahan, Alison E; Chan, Ying N; Lai, Jennifer I; Vaccari, Monica; Rao, Mangala; Franchini, Genoveffa; Alter, Galit; Ackerman, Margaret E

    2015-10-01

    Glycosylation of the Fc domain is an important driver of antibody effector function. While assessment of antibody glycoform compositions observed across total plasma IgG has identified differences associated with a variety of clinical conditions, in many cases it is the glycosylation state of only antibodies against a specific antigen or set of antigens that may be of interest, for example, in defining the potential effector function of antibodies produced during disease or after vaccination. Historically, glycoprofiling such antigen-specific antibodies in clinical samples has been challenging due to their low prevalence, the high sample requirement for most methods of glycan determination, and the lack of high-throughput purification methods. New methods of glycoprofiling with lower sample requirements and higher throughput have motivated the development of microscale and automatable methods for purification of antigen-specific antibodies from polyclonal sources such as clinical serum samples. In this work, we present a robot-compatible 96-well plate-based method for purification of antigen-specific antibodies, suitable for such population level glycosylation screening. We demonstrate the utility of this method across multiple antibody sources, using both purified plasma IgG and plasma, and across multiple different antigen types, with enrichment factors greater than 1000-fold observed. Using an on-column IdeS protease treatment, we further describe staged release of Fc and Fab domains, allowing for glycoprofiling of each domain. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Antibody-based immunotherapy for malignant glioma

    PubMed Central

    Gedeon, Patrick C.; Riccione, Katherine A.; Fecci, Peter E.; Sampson, John H.

    2014-01-01

    Conventional therapy for malignant glioma (MG) fails to specifically eliminate tumor cells, resulting in toxicity that limits therapeutic efficacy. In contrast, antibody-based immunotherapy utilizes the immune system to eliminate tumor cells with exquisite specificity. Increased understanding of the pathobiology of MG and the profound immunosuppression present among patients with MG has revealed several biologic targets amenable to antibody-based immunotherapy. Novel antibody engineering techniques allow for the production of fully human antibodies or antibody fragments with vastly reduced antigen-binding dissociation constants, increasing safety when used clinically as therapeutics. In this report, we summarize the use of antibody-based immunotherapy for MG. Approaches currently under investigation include the use of antibodies or antibody fragments to: 1) redirect immune effector cells to target tumor mutations, 2) inhibit immunosuppressive signals and thereby stimulate an immunological response against tumor cells, and 3) provide co-stimulatory signals to evoke immunologic targeting of tumor cells. These approaches demonstrate highly compelling safety and efficacy for the treatment of MG, providing a viable adjunct to current standard-of-care therapy for MG. PMID:25173142

  20. Antibody gene transfer for HIV immunoprophylaxis.

    PubMed

    Balazs, Alejandro B; West, Anthony P

    2013-01-01

    Antibody gene transfer, which involves the delivery of genes that encode potent, broadly neutralizing antibodies to human immunodeficiency virus (HIV), is a promising new strategy for preventing HIV infection. A satellite symposium at the AIDS Vaccine 2012 conference brought together many of the groups working in this field.

  1. 8th Annual European Antibody Congress 2012

    PubMed Central

    Beck, Alain; Carter, Paul J.; Gerber, Hans-Peter; Lugovskoy, Alexey A.; Wurch, Thierry; Junutula, Jagath R.; Kontermann, Roland E; Mabry, Robert

    2013-01-01

    The 8th European Antibody Congress (EAC), organized by Terrapin Ltd., was again held in Geneva, Switzerland, following on the tradition established with the 4th EAC. The new agenda format for 2012 included three parallel tracks on: (1) naked antibodies; (2) antibody drug conjugates (ADCs); and (3) bispecific antibodies and alternative scaffolds. The meeting started and closed with three plenary lectures to give common background and to share the final panel discussion and conclusions. The two day event included case studies and networking for nearly 250 delegates who learned of the latest advances and trends in the global development of antibody-based therapeutics. The monoclonal antibody track was focused on understanding the structure-function relationships, optimization of antibody design and developability, and processes that allow better therapeutic candidates to move through the clinic. Discussions on novel target identification and validation were also included. The ADC track was dedicated to evaluation of the ongoing success of the established ADC formats alongside the rise of the next generation drug-conjugates. The bispecific and alternative scaffold track was focused on taking stock of the multitude of bispecific formats being investigated and gaining insight into recent innovations and advancements. Mechanistic understanding, progression into the clinic and the exploration of multispecifics, redirected T cell killing and alternative scaffolds were extensively discussed. In total, nearly 50 speakers provided updates of programs related to antibody research and development on-going in the academic, government and commercial sectors. PMID:23493119

  2. IBC’s 23rd Annual Antibody Engineering, 10th Annual Antibody Therapeutics International Conferences and the 2012 Annual Meeting of The Antibody Society

    PubMed Central

    Klöhn, Peter-Christian; Wuellner, Ulrich; Zizlsperger, Nora; Zhou, Yu; Tavares, Daniel; Berger, Sven; Zettlitz, Kirstin A.; Proetzel, Gabriele; Yong, May; Begent, Richard H.J.; Reichert, Janice M

    2013-01-01

    The 23rd Annual Antibody Engineering, 10th Annual Antibody Therapeutics international conferences, and the 2012 Annual Meeting of The Antibody Society, organized by IBC Life Sciences with contributions from The Antibody Society and two Scientific Advisory Boards, were held December 3–6, 2012 in San Diego, CA. The meeting drew over 800 participants who attended sessions on a wide variety of topics relevant to antibody research and development. As a prelude to the main events, a pre-conference workshop held on December 2, 2012 focused on intellectual property issues that impact antibody engineering. The Antibody Engineering Conference was composed of six sessions held December 3–5, 2012: (1) From Receptor Biology to Therapy; (2) Antibodies in a Complex Environment; (3) Antibody Targeted CNS Therapy: Beyond the Blood Brain Barrier; (4) Deep Sequencing in B Cell Biology and Antibody Libraries; (5) Systems Medicine in the Development of Antibody Therapies/Systematic Validation of Novel Antibody Targets; and (6) Antibody Activity and Animal Models. The Antibody Therapeutics conference comprised four sessions held December 4–5, 2012: (1) Clinical and Preclinical Updates of Antibody-Drug Conjugates; (2) Multifunctional Antibodies and Antibody Combinations: Clinical Focus; (3) Development Status of Immunomodulatory Therapeutic Antibodies; and (4) Modulating the Half-Life of Antibody Therapeutics. The Antibody Society’s special session on applications for recording and sharing data based on GIATE was held on December 5, 2012, and the conferences concluded with two combined sessions on December 5–6, 2012: (1) Development Status of Early Stage Therapeutic Antibodies; and (2) Immunomodulatory Antibodies for Cancer Therapy. PMID:23575266

  3. Anti-miroestrol polyclonal antibodies: a comparison of immunogen preparations used to obtain desired antibody properties.

    PubMed

    Kitisripanya, Tharita; Jutathis, Kamonthip; Inyai, Chadathorn; Komaikul, Jukrapun; Udomsin, Orapin; Yusakul, Gorawit; Tanaka, Hiroyuki; Putalun, Waraporn

    2016-04-01

    Immunogen quality is one important factor that contributes to desirable antibody characteristics. Highly specific antibodies against miroestrol can be used to develop a quality control immunoassay for Pueraria candollei products. In this study, we investigated how various immunogen preparations affect antibody properties. The results show that immunogen prepared using the Mannich reaction provides antibodies with higher specificity and sensitivity against miroestrol than immunogen prepared with the periodate reaction. The results suggest the Mannich reaction maintains the original structure of miroestrol and generates useful antibodies for developing immunoassays.

  4. Trends in Malignant Glioma Monoclonal Antibody Therapy

    PubMed Central

    Chekhonin, Ivan; Gurina, Olga

    2015-01-01

    Although new passive and active immunotherapy methods are emerging, unconjugated monoclonal antibodies remain the only kind of biological preparations approved for high-grade glioma therapy in clinical practice. In this review, we combine clinical and experimental data discussion. As antiangiogenic therapy is the standard of care for recurrent glioblastoma multiforme (GBM), we analyze major clinical trials and possible therapeutic combinations of bevacizumab, the most common monoclonal antibody to vascular endothelial growth factor (VEGF). Another humanized antibody to gain recognition in GBM is epidermal growth factor (EGFR) antagonist nimotuzumab. Other antigens (VEGF receptor, platelet-derived growth factor receptor, hepatocyte growth factor and c-Met system) showed significance in gliomas and were used to create monoclonal antibodies applied in different malignant tumors. We assess the role of genetic markers (isocitrate dehydrogenase, O6-methylguanine-DNA methyltransnsferase) in GBM treatment outcome prediction. Besides antibodies studied in clinical trials, we focus on perspective targets and briefly list other means of passive immunotherapy.

  5. [Auto-antibodies in liver disease].

    PubMed

    Montaño Loza, Aldo J; Angulo, Paul

    2007-01-01

    Autoantibodies are a nonpathogenic manifestation of immune reactivity that may occur in acute and chronic liver diseases. Autoantibodies are the consequence rather than the cause of liver injury, and they can be used as diagnostic tools rather than etiologic markers. Conventional autoantibodies used in the categorization of liver disease are antinuclear antibodies, smooth muscle antibodies, antibodies to liver/kidney microsome type 1, antimitochondrial antibodies, and perinuclear antineutrophil cytoplasmic antibodies. However, the final diagnosis and the treatment strategies do not depend solely on the serological markers. Autoantibodies titles vary overtime and their behavior does not correlate with disease activity. Over-interpretation is the major pitfall in the clinical application of the serological results. Recognition and characterization of new autoantibodies is expected to improve the diagnostic precision, provide diagnostic parameters, and elucidate target autoantigens for the management of liver diseases.

  6. Conformational isomerism and the diversity of antibodies.

    PubMed Central

    Foote, J; Milstein, C

    1994-01-01

    The fact that one cell encodes a single antibody sequence does not necessarily mean that the resulting antibody folds into a single structure, although this is a common assumption. Here we challenge this view and suggest that many antibodies do not have a single conformation at the combining site. The basis for this proposal comes from the kinetic analysis of a set of murine hybridomas derived from defined stages of the immune response to 2-phenyl-5-oxazolone (Ox). Among them we have identified three antibodies that exhibit complex hapten-binding kinetics. We observed biphasic or triphasic reactions in stopped-flow fluorescence experiments, indicating that ligand binding involved isomerization, as well as associative steps. The existence of an equilibrium between at least two antibody conformations, with ligands binding preferentially to one form, was deduced from the variation with hapten concentration of the apparent rate of each phase. PMID:7937957

  7. CDR2 antigen and Yo antibodies.

    PubMed

    Totland, Cecilie; Aarskog, Nina K; Eichler, Tilo W; Haugen, Mette; Nøstbakken, Jane K; Monstad, Sissel E; Salvesen, Helga B; Mørk, Sverre; Haukanes, Bjørn I; Vedeler, Christian A

    2011-02-01

    Paraneoplastic cerebellar degeneration (PCD) is often associated with Yo antibodies that are directed against human cerebellar degeneration-related protein 2 (CDR2). Such antibodies may also be found in ovarian cancer patients without PCD. We studied if there was an association between Yo antibody production and differences in CDR2 cDNA sequence, mRNA or CDR2 expression in ovarian cancers. We found similar CDR2 cDNA sequence, mRNA and protein levels in primary ovarian cancers, with or without associated Yo antibodies. CDR2 was also present in other cancers, as well as in normal ovary tissue. The results suggest that Yo antibodies are not only related to the expression of CDR2 alone, but also to immune dysregulation.

  8. Antibodies in the treatment of aplastic anemia.

    PubMed

    Gómez-Almaguer, David; Jaime-Pérez, Jose Carlos; Ruiz-Arguelles, Guillermo J

    2012-04-01

    Antibodies have been the cornerstone of treatment of acquired aplastic anemia for more than 25 years. Treatment with antithymocyte globulin (ATG) is considered pivotal and the addition of cyclosporine improves the overall response rate. This antibody is heterogeneous and horse ATG is apparently more effective than rabbit ATG. Several issues remain unsolved in relation to the combination of ATG and cyclosporine: cost, toxicity and late clonal disorders. In recent years, alternative immunosuppressive therapy has been proposed and new antibodies have emerged: porcine ATG, alemtuzumab, daclizumab, and rituximab. Experience with these antibodies is limited to a few studies with alemtuzumab being the most promising, but the results are interesting and provocative. More studies are needed to find the perfect antibody.

  9. Monoclonal antibodies reacting with murine teratocarcinoma cells.

    PubMed Central

    Goodfellow, P N; Levinson, J R; Williams, V E; McDevitt, H O

    1979-01-01

    Monoclonal antibodies were produced in vitro by fusing mouse myeloma cells with spleen cells from a rat immunized with the C3H mouse teratocarcinoma C86-S1. After the fusion two clones were chosen for further analysis. The first clone, 3C4-10, produced an antibody recognizing an antigen with a distribution restricted to teratocarcinoma cell lines, an endoderm cell line, and a neuroblastoma. The second clone, 4A1-9, produced an antibody that reacted with all cultured murine cells tested and adult brain. Neither antibody reacted with preimplantation embryos. The 3C4-10 antibody recognized an antigen associated with proteins. The apparent molecular weight of the 3C4-10 antigen was greater than 100,000. PMID:284353

  10. Clearance of pathological antibodies using biomimetic nanoparticles

    PubMed Central

    Copp, Jonathan A.; Fang, Ronnie H.; Luk, Brian T.; Hu, Che-Ming J.; Gao, Weiwei; Zhang, Kang; Zhang, Liangfang

    2014-01-01

    Pathological antibodies have been demonstrated to play a key role in type II immune hypersensitivity reactions, resulting in the destruction of healthy tissues and leading to considerable morbidity for the patient. Unfortunately, current treatments present significant iatrogenic risk while still falling short for many patients in achieving clinical remission. In the present work, we explored the capability of target cell membrane-coated nanoparticles to abrogate the effect of pathological antibodies in an effort to minimize disease burden, without the need for drug-based immune suppression. Inspired by antibody-driven pathology, we used intact RBC membranes stabilized by biodegradable polymeric nanoparticle cores to serve as an alternative target for pathological antibodies in an antibody-induced anemia disease model. Through both in vitro and in vivo studies, we demonstrated efficacy of RBC membrane-cloaked nanoparticles to bind and neutralize anti-RBC polyclonal IgG effectively, and thus preserve circulating RBCs. PMID:25197051

  11. Radiolabeled antibodies for therapy of infectious diseases

    PubMed Central

    Dadachova, Ekaterina; Casadevall, Arturo

    2014-01-01

    Novel approaches to treatment of infectious diseases are urgently needed. This need has resulted in renewing the interest in antibodies for therapy of infectious diseases. Radioimmunotherapy (RIT) is a cancer treatment modality, which utilizes radiolabeled monoclonal antibodies (mAbs). During the last decade we have translated RIT into the field of experimental fungal, bacterial and HIV infections. In addition, successful proof of principle experiments with radiolabeled pan-antibodies that bind to antigens shared by major pathogenic fungi were performed in vitro. The armamentarium of pan-antibodies would result in reducing the dependence on microorganism-specific antibodies and thus would speed up the development of RIT of infections. We believe that the time is ripe for deploying RIT into the clinic to combat infectious diseases. PMID:25599011

  12. Luminex and antibody detection in kidney transplantation.

    PubMed

    Picascia, Antonietta; Infante, Teresa; Napoli, Claudio

    2012-06-01

    Preformed anti-human leukocyte antigen (HLA) antibodies have a negative effect on kidney transplantation outcome with an increased rejection rate and reduction in survival. Posttransplantation production of donor-specific anti-HLA antibodies is indicative of an active immune response and risk of transplantation rejection. For many years the primary technique for anti-HLA antibody detection was complement-dependent cytotoxicity (CDC), which has been integrated by solid-phase assays as HLA antigen-coated bead methods (Luminex). This new technological approach has allowed identification of anti-HLA antibodies, not detectable using conventional CDC method, in patients awaiting kidney transplantation. Moreover, use of Luminex technology has enabled better definition of acceptable or unacceptable antigens favoring transplantation in highly immunized patients. However, there are still many unresolved issues, including the clinical relevance of antibodies detected with this system.

  13. Monoclonal antibodies in chronic lymphocytic leukemia.

    PubMed

    Ferrajoli, Alessandra; Faderl, Stefan; Keating, Michael J

    2006-09-01

    Multiple options are now available for the treatment of chronic lymphocytic leukemia. Over the last 10 years, monoclonal antibodies have become an integral part of the management of this disease. Alemtuzumab has received approval for use in patients with fludarabine-refractory chronic lymphocytic leukemia. Rituximab has been investigated extensively in chronic lymphocytic leukemia both as a single agent and in combination with chemotherapy and other monoclonal antibodies. Epratuzumab and lumiliximab are newer monoclonal antibodies in the early phase of clinical development. This article will review the monoclonal antibodies more commonly used to treat chronic lymphocytic leukemia, the results obtained with monoclonal antibodies as single agents and in combination with chemotherapy, and other biological agents and newer compounds undergoing clinical trials.

  14. Edelman's view on the discovery of antibodies.

    PubMed

    Ribatti, Domenico

    2015-04-01

    Gerald M. Edelman began working to the structure of antibodies when joined as graduate student the laboratory of Henry Kunkel in 1958 at the "Rockefeller University" in New York, obtaining his doctorate in 1960. Edelman's focus on the structure of antibodies led to the 1972 Nobel Prize in Physiology or Medicine along with Rodney R. Porter. Edelman and Porter decided to approach the problem of antibodies structure by splitting. In 1959, Porter published a report in which he used the enzyme papain to cleave the antibody molecule into three pieces of about 50,000 Da, corresponding to the two Fab (antigen-binding) and constant Fc (crystallizable) fragments. In the same year, Edelman showed that reduction of the disulfide bonds of antibodies in the presence of denaturizing agents led to dissociation of the molecule into smaller pieces, now known to be the light (L) and heavy (H) chains.

  15. Development and Detection of Kidd Antibodies.

    PubMed

    Sanford, Kimberly Williams; Bourikian, Seda; McClain, Aryn; Curtis, Kyle

    2015-01-01

    Kidd antibodies have a reputation for causing hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. We present a case of an untransfused male patient who developed anti-Kidd(a) (Jk(a)) antibodies after receiving an allogenic renal transplant. The formation of this antibody was associated with exposure to the Kidd antigen expressed on the tubular epithelium of the transplanted kidney. The 59-year-old white male patient had received a cadaveric renal transplant at our clinic and returned 5 years later with proteinuria and elevated serum creatinine levels, consistent with nephrotic syndrome. We review the expression of Kidd antigens and the development and detection of Kidd antibodies, and discuss the case reports from the literature of Kidd antibodies associated with kidney-graft rejection that suggest Kidd antigens play a role as a minor histocompatibility antigen.

  16. Antibodies to laminin in American cutaneous leishmaniasis.

    PubMed Central

    Avila, J L; Rojas, M; Rieber, M

    1984-01-01

    We found that serum samples from patients with different clinical forms of American cutaneous leishmaniasis (ACL) contained immunoglobulin G and immunoglobulin M antibodies which reacted with laminin but not with various other purified connective tissue components, such as collagen types I, III, IV, and V and fibronectin. Eighty-one percent of ACL patients had high antilaminin antibody levels, with a relationship existing between ACL ulcers and antibody levels. This was not, however, the case with patients having treated and healed ACL ulcers; only 34% of these patients had elevated antilaminin antibodies. Eighty-four percent of chronic Chagas' disease patients were also found to contain antilaminin antibodies that were limited to the immunoglobulin G class, but these were not detected in patients suffering from any of 11 other infectious diseases. PMID:6418660

  17. Antibody-Mediated Lung Transplant Rejection

    PubMed Central

    Hachem, Ramsey

    2012-01-01

    Antibody-mediated rejection after lung transplantation remains enigmatic. However, emerging evidence over the past several years suggests that humoral immunity plays an important role in allograft rejection. Indeed, the development of donor-specific antibodies after transplantation has been identified as an independent risk factor for acute cellular rejection and bronchiolitis obliterans syndrome. Furthermore, cases of acute antibody-mediated rejection resulting in severe allograft dysfunction have been reported, and these demonstrate that antibodies can directly injure the allograft. However, the incidence and toll of antibody-mediated rejection are unknown because there is no widely accepted definition and some cases may be unrecognized. Clearly, humoral immunity has become an important area for research and clinical investigation. PMID:23002428

  18. Antibody production by in vivo RNA transfection.

    PubMed

    Romani, Bizhan; Kavyanifard, Amirarsalan; Allahbakhshi, Elham

    2017-09-07

    Monoclonal antibodies have a variety of applications in research and medicine. Here, we report development of a new method for production of monoclonal antibodies. Our method relies on in vivo RNA transfection rather than peptide vaccination. We took advantage of RNA transcripts complexed with DOTMA and DOPE lipids to transfect mice. Intravenous administration of our RNA vaccine to mice resulted in expression of the antigenic peptides by splenic dendritic cells and detection of the antigens in the serum. The RNA vaccine stimulated production of specific antibodies against the RNA-encoded peptides. We produced monoclonal antibodies against viral, bacterial, and human antigens. In addition, we showed that our RNA vaccine stimulated humoral immunity and rescued mice infected with influenza A virus. Our method could be used as an efficient tool to generate monoclonal antibodies and to stimulate humoral immunity for research and medical purposes.

  19. Antibody-based resistance to plant pathogens.

    PubMed

    Schillberg, S; Zimmermann, S; Zhang, M Y; Fischer, R

    2001-01-01

    Plant diseases are a major threat to the world food supply, as up to 15% of production is lost to pathogens. In the past, disease control and the generation of resistant plant lines protected against viral, bacterial or fungal pathogens, was achieved using conventional breeding based on crossings, mutant screenings and backcrossing. Many approaches in this field have failed or the resistance obtained has been rapidly broken by the pathogens. Recent advances in molecular biotechnology have made it possible to obtain and to modify genes that are useful for generating disease resistant crops. Several strategies, including expression of pathogen-derived sequences or anti-pathogenic agents, have been developed to engineer improved pathogen resistance in transgenic plants. Antibody-based resistance is a novel strategy for generating transgenic plants resistant to pathogens. Decades ago it was shown that polyclonal and monoclonal antibodies can neutralize viruses, bacteria and selected fungi. This approach has been improved recently by the development of recombinant antibodies (rAbs). Crop resistance can be engineered by the expression of pathogen-specific antibodies, antibody fragments or antibody fusion proteins. The advantages of this approach are that rAbs can be engineered against almost any target molecule, and it has been demonstrated that expression of functional pathogen-specific rAbs in plants confers effective pathogen protection. The efficacy of antibody-based resistance was first shown for plant viruses and its application to other plant pathogens is becoming more established. However, successful use of antibodies to generate plant pathogen resistance relies on appropriate target selection, careful antibody design, efficient antibody expression, stability and targeting to appropriate cellular compartments.

  20. Vector-Mediated In Vivo Antibody Expression.

    PubMed

    Schnepp, Bruce C; Johnson, Philip R

    2014-08-01

    This article focuses on a novel vaccine strategy known as vector-mediated antibody gene transfer, with a particular focus on human immunodeficiency virus (HIV). This strategy provides a solution to the problem of current vaccines that fail to generate neutralizing antibodies to prevent HIV-1 infection and AIDS. Antibody gene transfer allows for predetermination of antibody affinity and specificity prior to "immunization" and avoids the need for an active humoral immune response against the HIV envelope protein. This approach uses recombinant adeno-associated viral (rAAV) vectors, which have been shown to transduce muscle with high efficiency and direct the long-term expression of a variety of transgenes, to deliver the gene encoding a broadly neutralizing antibody into the muscle. Following rAAV vector gene delivery, the broadly neutralizing antibodies are endogenously synthesized in myofibers and passively distributed to the circulatory system. This is an improvement over classical passive immunization strategies that administer antibody proteins to the host to provide protection from infection. Vector-mediated gene transfer studies in mice and monkeys with anti-HIV and simian immunodeficiency virus (SIV)-neutralizing antibodies demonstrated long-lasting neutralizing activity in serum with complete protection against intravenous challenge with virulent HIV and SIV. These results indicate that existing potent anti-HIV antibodies can be rapidly moved into the clinic. However, this methodology need not be confined to HIV. The general strategy of vector-mediated antibody gene transfer can be applied to other difficult vaccine targets such as hepatitis C virus, malaria, respiratory syncytial virus, and tuberculosis.

  1. Measurement of tumour reactive antibody and antibody conjugate by competition, quantitated by flow cytofluorimetry.

    PubMed

    Robins, R A; Laxton, R R; Garnett, M; Price, M R; Baldwin, R W

    1986-06-24

    Binding of unlabelled monoclonal antibody preparations has been assessed by competition at saturation with fluorochrome labelled homologous antibody for binding to antigen bearing target cells. The extent of competition was measured by quantitative flow cytofluorimetry, and simple mathematical procedures have been developed to allow the interpretation of competition data in terms of antibody binding activity. In the system studied, non-specific (non-competitive) fluorescence was minimal, but an iterative method to calculate its contribution to the measured signal is given. This approach has the advantage that the antibody preparation to be tested does not need to be labelled or modified; this is particularly important when evaluating the binding activity of therapeutic antibody conjugates. Comparison with a well characterized standard antibody preparation provides a rapid, sensitive and accurate quality control procedure. This test is also simple to perform, requiring only the mixing of labelled and unlabelled antibodies with target cells, a single incubation, followed by analysis without washing of the target cells.

  2. High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells.

    PubMed

    Jäger, Volker; Büssow, Konrad; Wagner, Andreas; Weber, Susanne; Hust, Michael; Frenzel, André; Schirrmann, Thomas

    2013-06-26

    The demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable as immunoglobulins (Ig)G in many immunoassays. In contrast, the bivalent scFv-Fc antibody format shares many properties with IgG and has a very high application compatibility. In this study transient expression of scFv-Fc antibodies in human embryonic kidney (HEK) 293 cells was optimized. Production levels of 10-20 mg/L scFv-Fc antibody were achieved in adherent HEK293T cells. Employment of HEK293-6E suspension cells expressing a truncated variant of the Epstein Barr virus (EBV) nuclear antigen (EBNA) 1 in combination with production under serum free conditions increased the volumetric yield up to 10-fold to more than 140 mg/L scFv-Fc antibody. After vector optimization and process optimization the yield of an scFv-Fc antibody and a cytotoxic antibody-RNase fusion protein further increased 3-4-fold to more than 450 mg/L. Finally, an entirely new mammalian expression vector was constructed for single step in frame cloning of scFv genes from antibody phage display libraries. Transient expression of more than 20 different scFv-Fc antibodies resulted in volumetric yields of up to 600 mg/L and 400 mg/L in average. Transient production of recombinant scFv-Fc antibodies in HEK293-6E in combination with optimized vectors and fed batch shake flasks cultivation is efficient and robust, and integrates well into a high-throughput recombinant antibody generation pipeline.

  3. Radiohalogenated half-antibodies and maleimide intermediate therefor

    DOEpatents

    Kassis, A.I.; Khawli, L.A.

    1991-02-19

    N-(m-radiohalophenyl) maleimide can be conjugated with a reduced antibody having a mercapto group to provide a radiolabeled half-antibody having immunological specific binding characteristics of whole antibody. No Drawings

  4. Radiohalogenated half-antibodies and maleimide intermediate therefor

    DOEpatents

    Kassis, Amin I.; Khawli, Leslie A.

    1991-01-01

    N-(m-radiohalophenyl) maleimide can be conjugated with a reduced antibody having a mercapto group to provide a radiolabelled half-antibody having immunological specific binding characteristics of whole antibody.

  5. Principles and application of antibody libraries for infectious diseases.

    PubMed

    Lim, Bee Nar; Tye, Gee Jun; Choong, Yee Siew; Ong, Eugene Boon Beng; Ismail, Asma; Lim, Theam Soon

    2014-12-01

    Antibodies have been used efficiently for the treatment and diagnosis of many diseases. Recombinant antibody technology allows the generation of fully human antibodies. Phage display is the gold standard for the production of human antibodies in vitro. To generate monoclonal antibodies by phage display, the generation of antibody libraries is crucial. Antibody libraries are classified according to the source where the antibody gene sequences were obtained. The most useful library for infectious diseases is the immunized library. Immunized libraries would allow better and selective enrichment of antibodies against disease antigens. The antibodies generated from these libraries can be translated for both diagnostic and therapeutic applications. This review focuses on the generation of immunized antibody libraries and the potential applications of the antibodies derived from these libraries.

  6. Antiendomysial antibodies in Berger's disease.

    PubMed

    Pierucci, Alessandro; Fofi, Claudia; Bartoli, Benedetta; Simonetti, Bianca Maria; Pecci, Gabriella; Sabbatella, Luigi; Di Tola, Marco; Greco, Rosita; Anania, Maria Cristina; Picarelli, Antonio

    2002-06-01

    The finding of increased levels of immunoglobulin A (IgA) against food antigens in patients with IgA nephropathy prompted the hypothesis of an association between IgA nephropathy and celiac disease (CD). Attention was initially directed to antigliadin antibodies, then to IgA antiendomysial antibodies (IgA-EMA). IgG1-EMA have been found in patients with CD with IgA-EMA-negative results. The presence of IgA- and IgG1-EMA was investigated in 36 patients with IgA nephropathy, 15 patients with other primary glomerulonephritis, and 15 patients with lupus nephritis. IgA-EMA and IgG1-EMA were detected by indirect immunofluorescence analysis. At the time of renal biopsy, the following factors were evaluated: history of macroscopic hematuria, serum creatinine level, urinalysis, 24-hour proteinuria, blood pressure, and histological classification of IgA nephropathy. Sixteen of 36 patients with IgA nephropathy (44.4%) showed EMA positivity. Among patients with positive EMA, 12 patients (75%) were IgG1-EMA positive, 2 patients (12.5%) were IgA-EMA positive, and 2 patients (12.5%) were positive for both isotypes. No significant differences were observed between the two groups (EMA positive versus EMA negative) concerning age, serum creatinine level, macroscopic hematuria, blood pressure, 24-hour proteinuria, or degree of renal histological involvement. IgA- and IgG1-EMA were not detected in patients with other primary nephropathies or lupus nephritis. These results, based on the finding of IgG1-EMA, suggest a common pathogenetic pathway for CD and IgA nephropathy. On this basis, the presence of IgG1-EMA and/or IgA-EMA should be investigated in patients with IgA nephropathy. Furthermore, the role of a gluten-free diet in the natural history of IgA nephropathy, at least in EMA-positive patients, needs to be ascertained.

  7. Antibody Engineering & Therapeutics, the annual meeting of The Antibody Society December 7-10, 2015, San Diego, CA, USA.

    PubMed

    Pauthner, Matthias; Yeung, Jenny; Ullman, Chris; Bakker, Joost; Wurch, Thierry; Reichert, Janice M; Lund-Johansen, Fridtjof; Bradbury, Andrew R M; Carter, Paul J; Melis, Joost P M

    2016-01-01

    The 26th Antibody Engineering & Therapeutics meeting, the annual meeting of The Antibody Society united over 800 participants from all over the world in San Diego from 6-10 December 2015. The latest innovations and advances in antibody research and development were discussed, covering a myriad of antibody-related topics by more than 100 speakers, who were carefully selected by The Antibody Society. As a prelude, attendees could join the pre-conference training course focusing, among others, on the engineering and enhancement of antibodies and antibody-like scaffolds, bispecific antibody engineering and adaptation to generate chimeric antigen receptor constructs. The main event covered 4 d of scientific sessions that included antibody effector functions, reproducibility of research and diagnostic antibodies, new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new technologies and applications for bispecific antibodies, antibody therapeutics for non-cancer and orphan indications, antibodies to harness the cellular immune system, building comprehensive IgVH-gene repertoires through discovering, confirming and cataloging new germline IgVH genes, and overcoming resistance to clinical immunotherapy. The Antibody Society's special session focused on "Antibodies to watch" in 2016. Another special session put the spotlight on the limitations of the new definitions for the assignment of antibody international nonproprietary names introduced by the World Health Organization. The convention concluded with workshops on computational antibody design and on the promise and challenges of using next-generation sequencing for antibody discovery and engineering from synthetic and in vivo libraries.

  8. Antibodies as predictors of complex autoimmune diseases.

    PubMed

    Vojdani, A

    2008-01-01

    Emerging evidence has suggested environmental factors such as infections and xenobiotics and some dietary proteins and peptides in the pathogenesis of many autoimmune diseases. Considering the fact that autoantibodies can often be detected prior to the onset of a disease, in this study an enzyme immunoassay was used for measurement of antibodies against different highly purified antigens or synthetic peptides originating not only from human tissue, but also from cross-reactive epitopes of infectious agents, dietary proteins and xenobiotics. The measurement of antibodies against a panel of antigens allows for identification of patterns or antibody signatures, rather than just one or two markers of autoimmunity, thus establishing the premise for increased sensitivity and specificity of prediction, as well as positive predictive values. This panel of different autoantibodies was applied to 420 patients with different autoimmune diseases, including pernicious anemia, celiac disease, thyroiditis, lupus, rheumatoid arthritis, osteoarthritis, Addison's disease, type 1 diabetes, cardiovascular disease and autoimmunity, which are presented in this article. In all cases, the levels of these antibodies were significantly elevated in patients versus controls. Antibody patterns related to neuroautoimmune disorders, cancer, and patients with somatic hypermutation will be shown in a subsequent article. We believe that this novel 96 antigen-specific autoantibody or predictive antibody screen should be studied for its incorporation into routine medical examinations. Clinicians should be aware that the detection of antibodies should not automatically mean that a patient will definitely become ill, but would rather give a percentage of risk for autoimmune disease over subsequent months or years.

  9. Monoclonal antibodies in the treatment of cancer

    SciTech Connect

    Dillman, R.O.

    1984-01-01

    Potential uses of monoclonal antibodies in anti-cancer treatment include passive serotherapy, radioisotope conjugates, toxin-linked conjugates, and chemotherapy-monoclonal antibody conjugates. The bases for these applications have been founded in research with heterologous antisera, and in some cases with monoclonal antibodies in animal tumor models. Human trials with passive serotherapy have already begun in both hematopoietic and solid tumor malignancies. Promising results have been reported in cutaneous T cell lymphoma with anti-T cell monoclonal antibody, and in nodular lymphoma with anti-idiotype monoclonal antibody. Radioisotope conjugate work appears promising for imaging in both animals and humans, and this work will lay the foundation for possible therapeutic application of radio-immunotherapy. Toxin-linked conjugates are promising in vitro and may have application in autologous bone marrow transplantation. Research with chemotherapy conjugates is also underway. Preliminary results suggest that murine monoclonal antibodies will be well tolerated clinically except in the setting of circulating cells which bear the target antigen, where rapid infusions may be associated with intolerable side effects. In certain diseases, production of endogenous anti-mouse antibodies may also limit application. Advances in the technology for human-human hybridoma production may help solve some of these problems. 132 references.

  10. Glycosylation profiles of therapeutic antibody pharmaceuticals.

    PubMed

    Wacker, Christoph; Berger, Christoph N; Girard, Philippe; Meier, Roger

    2011-11-01

    Recombinant antibodies specific for human targets are often used as therapeutics and represent a major class of drug products. Their therapeutic efficacy depends on the formation of antibody complexes resulting in the elimination of a target molecule or the modulation of specific signalling pathways. The physiological effects of antibody therapeutics are known to depend on the structural characteristics of the antibody molecule, specifically on the glycosylation which is the result of posttranslational modifications. Hence, production of therapeutic antibodies with a defined and consistent glycoform profile is needed which still remains a considerable challenge to the biopharmaceutical industry. To provide an insight into the industries capability to control their manufacturing process and to provide antibodies of highest quality, we conducted a market surveillance study and compared major oligosaccharide profiles of a number of monoclonal antibody pharmaceuticals sampled on the Swiss market. Product lot-to-lot variability was found to be generally low, suggesting that a majority of manufacturers have implemented high quality standards in their production processes. However, proportions of G0, G1 and G2 core-fucosylated chains derived from different products varied considerably and showed a bias towards the immature agalactosidated G0 form. Interestingly, differences in glycosylation caused by the production cell type seem to be of less importance compared with process related parameters such as cell growth.

  11. Glycosylation of plant produced human antibodies.

    PubMed

    Kallolimath, Somanath; Steinkellner, Herta

    2015-12-23

    Human immunoglobulins circulate as highly heterogeneously glycosylated mixture of otherwise homogeneous protein backbones. A series of studies, mainly on IgG, have unequivocally proven that antibodies modulate their effector function through sugars present in the Fc domain. However, our limited technology in producing complex proteins such as antibodies, with defined glycan structures hamper in depths studies. This review introduces a plant based expression platform enabling engineering of antibody glycans. The procedure is based on the simultaneous delivery of appropriate constructs, carrying cDNAs of target proteins (e.g. heavy and light chain of antibodies) in combination with human glycosylation enzymes into plant leaves. Harvesting of recombinant proteins one week post construct delivery allows high speed and flexibility. Major achievements include the production of functional active slialylated pentameric IgMs in tobacco leaves. The system provides a viable approach to the generation of antibodies with defined glycoforms on demand, contributing to studies on antibody glycans and the development of novel antibody based drugs.

  12. Baculovirus display of functional antibody Fab fragments.

    PubMed

    Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

    2015-08-01

    The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

  13. Antibody binding loop insertions as diversity elements

    PubMed Central

    Kiss, Csaba; Fisher, Hugh; Pesavento, Emanuele; Dai, Minghua; Valero, Rosa; Ovecka, Milan; Nolan, Rhiannon; Phipps, M. Lisa; Velappan, Nileena; Chasteen, Leslie; Martinez, Jennifer S.; Waldo, Geoffrey S.; Pavlik, Peter; Bradbury, Andrew R.M.

    2006-01-01

    In the use of non-antibody proteins as affinity reagents, diversity has generally been derived from oligonucleotide-encoded random amino acids. Although specific binders of high-affinity have been selected from such libraries, random oligonucleotides often encode stop codons and amino acid combinations that affect protein folding. Recently it has been shown that specific antibody binding loops grafted into heterologous proteins can confer the specific antibody binding activity to the created chimeric protein. In this paper, we examine the use of such antibody binding loops as diversity elements. We first show that we are able to graft a lysozyme-binding antibody loop into green fluorescent protein (GFP), creating a fluorescent protein with lysozyme-binding activity. Subsequently we have developed a PCR method to harvest random binding loops from antibodies and insert them at predefined sites in any protein, using GFP as an example. The majority of such GFP chimeras remain fluorescent, indicating that binding loops do not disrupt folding. This method can be adapted to the creation of other nucleic acid libraries where diversity is flanked by regions of relative sequence conservation, and its availability sets the stage for the use of antibody loop libraries as diversity elements for selection experiments. PMID:17023486

  14. Progress towards recombinant anti-infective antibodies

    PubMed Central

    Pai, Jennifer C.; Sutherland, Jamie N.; Maynard, Jennifer A.

    2009-01-01

    The global market for monoclonal antibody therapeutics reached a total of $11.2 billion in 2004, with an impressive 42% growth rate over the previous five years and is expected to reach ~$34 billion by 2010. Coupled with this growth are stream-lined product development, production scale-up and regulatory approval processes for the highly conserved antibody structure. While only one of the 21 current FDA-approved antibodies, and one of the 38 products in advanced clinical trials target infectious diseases, there is increasing academic, government and commercial interest in this area. Synagis, an antibody neutralizing respiratory syncitial virus (RSV), garnered impressive sales of $1.1 billion in 2006 in spite of its high cost and undocumented effects on viral titres in human patients. The success of anti-RSV passive immunization has motivated the continued development of anti-infectives to treat a number of other infectious diseases, including those mediated by viruses, toxins and bacterial/fungal cells. Concurrently, advances in antibody technology suggest that cocktails of several monoclonal antibodies with unique epitope specificity or single monoclonal antibodies with broad serotype specificity may be the most successful format. Recent patents and patent applications in these areas will be discussed as predictors of future anti-infective therapeutics. PMID:19149692

  15. HIV antibodies for treatment of HIV infection.

    PubMed

    Margolis, David M; Koup, Richard A; Ferrari, Guido

    2017-01-01

    The bar is high to improve on current combination antiretroviral therapy (ART), now highly effective, safe, and simple. However, antibodies that bind the HIV envelope are able to uniquely target the virus as it seeks to enter new target cells, or as it is expressed from previously infected cells. Furthermore, the use of antibodies against HIV as a therapeutic may offer advantages. Antibodies can have long half-lives, and are being considered as partners for long-acting antiretrovirals for use in therapy or prevention of HIV infection. Early studies in animal models and in clinical trials suggest that such antibodies can have antiviral activity but, as with small-molecule antiretrovirals, the issues of viral escape and resistance will have to be addressed. Most promising, however, are the unique properties of anti-HIV antibodies: the potential ability to opsonize viral particles, to direct antibody-dependent cellular cytotoxicity (ADCC) against actively infected cells, and ultimately the ability to direct the clearance of HIV-infected cells by effector cells of the immune system. These distinctive activities suggest that HIV antibodies and their derivatives may play an important role in the next frontier of HIV therapeutics, the effort to develop treatments that could lead to an HIV cure. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  16. Next generation of antibody therapy for cancer

    PubMed Central

    Zhu, Zhenping; Yan, Li

    2011-01-01

    Monoclonal antibodies (mAbs) have become a major class of therapeutic agents providing effective alternatives to treating various human diseases. To date, 15 mAbs have been approved by regulatory agencies in the world for clinical use in oncology indications. The selectivity and specificity, the unique pharmacokinetics, and the ability to engage and activate the host immune system differentiate these biologics from traditional small molecule anticancer drugs. mAb-based regimens have brought clinical benefits, including improvements in overall survival, to patients with a variety of cancers. Many challenges still remain, however, to fully realize the potential of these new medicines. With our further understanding of cancer biology, mechanism of antibody action, and advancement of antibody engineering technologies, many novel antibody formats or antibody-derived molecules are emerging as promising new generation therapeutics. Carefully designed and engineered, they retain the advantage of specificity and selectivity of original antibodies, but in the meantime acquire additional special features such as improved pharmacokinetics, increased selectivity, and enhanced anticancer efficacy. Promising clinical results are being generated with these newly improved antibody-based therapeutics. PMID:21527062

  17. 21 CFR 866.5090 - Antimitochondrial antibody immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... immunochemical techniques the antimitochondrial antibodies in human serum. The measurements aid in the diagnosis of diseases that produce a spectrum of autoantibodies (antibodies produced against the body's...

  18. Combinatorial antibody libraries: new advances, new immunological insights.

    PubMed

    Lerner, Richard A

    2016-08-01

    Immunochemists have become quite proficient in engineering existing antibody molecules to control their pharmacological properties. However, in terms of generating new antibodies, the combinatorial antibody library has become a central feature of modern immunochemistry. These libraries are essentially an immune system in a test tube and enable the selection of antibodies without the constraints of whole animal or cell-based systems. This Review provides an overview of how antibody libraries are constructed and discusses what can be learnt from these synthetic systems. In particular, the Review focuses on new biological insights from antibody libraries - such as the concept of 'SOS antibodies' - and the growing use of intracellular antibodies to perturb cellular functions.

  19. Distinct Therapeutic Mechanisms of Tau Antibodies

    PubMed Central

    Funk, Kristen E.; Mirbaha, Hilda; Jiang, Hong; Holtzman, David M.; Diamond, Marc I.

    2015-01-01

    Tauopathies are neurodegenerative diseases characterized by accumulation of Tau amyloids, and include Alzheimer disease and certain frontotemporal dementias. Trans-neuronal propagation of amyloid mediated by extracellular Tau may underlie disease progression. Consistent with this, active and passive vaccination studies in mouse models reduce pathology, although by unknown mechanisms. We previously reported that intracerebroventricular administration of three anti-Tau monoclonal antibodies (HJ8.5, HJ9.3, and HJ9.4) reduces pathology in a model overexpressing full-length mutant (P301S) human Tau. We now study effects of these three antibodies and a negative control antibody (HJ3.4) on Tau aggregate uptake into BV2 microglial-like cells and primary neurons. Antibody-independent Tau uptake into BV2 cells was blocked by heparin, consistent with a previously described role for heparan sulfate proteoglycans. Two therapeutic antibodies (HJ8.5 and HJ9.4) promoted uptake of full-length Tau fibrils into microglia via Fc receptors. Surprisingly, HJ9.3 promoted uptake of fibrils composed of the Tau repeat domain or Alzheimer disease-derived Tau aggregates, but failed to influence full-length recombinant Tau fibrils. Size fractionation of aggregates showed that antibodies preferentially promote uptake of larger oligomers (n ≥∼20-mer) versus smaller oligomers (n ∼10-mer) or monomer. No antibody inhibited uptake of full-length recombinant fibrils into primary neurons, but HJ9.3 blocked neuronal uptake of Tau repeat domain fibrils and Alzheimer disease-derived Tau. Antibodies thus have multiple potential mechanisms, including clearance via microglia and blockade of neuronal uptake. However these effects are epitope- and aggregate size-dependent. Establishing specific mechanisms of antibody activity in vitro may help in design and optimization of agents that are more effective in vivo. PMID:26126828

  20. Labeling of monoclonal antibodies with radionuclides

    SciTech Connect

    Bhargava, K.K.; Acharya, S.A. )

    1989-07-01

    Antibodies, specifically monoclonal antibodies, are potentially very useful and powerful carriers of therapeutic agents to target tissues and diagnostic agents. The loading or charging of antibodies with agents, especially radiotracers, is reviewed here. The choice of radioisotope for immunodetection and/or immunotherapy is based on its availability, half-life, nature of the radiation emitted, and the metabolic pathways of the radionuclide in the body. Most important of all are the derivatization techniques available for labeling the antibody with the given radionuclide. Isotopes of iodine and divalent metal ions are the most commonly used radionuclides. Antibodies labeled with iodine at tyrosine residues are metabolized rapidly in vivo. This leads to the incorporation of metabolized radioactive iodine into various tissues, mainly the thyroid gland and stomach, and to the accumulation of high levels of circulating iodine in the blood, which masks tumor uptake considerably. To overcome these limitations, the use of iodohippurate as an iodine-anchoring molecule to the protein should be considered. When divalent or multivalent metal ions are used as the preferred radionuclide, bifunctional chelating reagents such as EDTA or DTPA are first coupled to the protein or antibody. These chelating molecules are attached to the protein by formation of an isopeptide linkage between the carboxylate of the chelating reagent and the amino group of the protein. Several procedures are available to generate the isopeptide linkage. When the anchoring of the chelating agent through isopeptide linkage results in the inactivation of the antibody, periodate oxidation of the carbohydrate moiety of the antibody, followed by reductive coupling of chelator, could be considered as an alternative. There is still a need for better, simpler, and more direct methods for labeling antibodies with radionuclides. 78 references.

  1. Antibodies Against Three Forms of Urokinase

    NASA Technical Reports Server (NTRS)

    Morrison, Dennis R.; Atassi, M. Zouhair

    2007-01-01

    Antibodies that bind to preselected regions of the urokinase molecule have been developed. These antibodies can be used to measure small quantities of each of three molecular forms of urokinase that could be contained in microsamples or conditioned media harvested from cultures of mammalian cells. Previously available antibodies and assay techniques do not yield both clear distinctions among, and measurements of, all three forms. Urokinase is a zymogen that is synthesized in a single-chain form, called ScuPA, which is composed of 411 amino acid residues (see figure). ScuPA has very little enzyme activity, but it can be activated in two ways: (1) by cleavage of the peptide bond lysine 158/isoleucine 159 and the loss of lysine 158 to obtain the high molecular-weight (HMW) form of the enzyme or (2) by cleavage of the bond lysine 135/lysine 136 to obtain the low-molecular-weight (LMW) form of the enzyme. The antibodies in question were produced in mice and rabbits by use of peptides as immunogens. The peptides were selected to obtain antibodies that bind to regions of ScuPA that include the lysine 158/isoleucine 159 and the lysine 135/lysine 136 bonds. The antibodies include monoclonal and polyclonal ones that yield indications as to whether either of these bonds is intact. The polyclonal antibodies include ones that preferentially bind to the HMW or LMW forms of the urokinase molecule. The monoclonal antibodies include ones that discriminate between the ScuPA and the HMW form. A combination of these molecular-specific antibodies will enable simultaneous assays of the ScuPA, HMW, and LMW forms in the same specimen of culture medium.

  2. Coeliac Disease-Associated Antibodies in Psoriasis

    PubMed Central

    Akbulut, Sabiye; Gür, Günes; Topal, Firdevs; Topal, Fatih Esad; Alli, Nuran; Saritas, Ülkü

    2013-01-01

    Background The possible relationship between psoriasis and coeliac disease (CD) has been attributed to the common pathogenic mechanisms of the two diseases and the presence of antigliadin antibodies in patients has been reported to increase the incidence of CD. Objective The aim of this report was to study CD-associated antibodies serum antigliadin antibody immunoglobulin (Ig)A, IgG, anti-endomysial antibody IgA and anti-transglutaminase antibody IgA and to demonstrate whether there is an increase in the frequency of those markers of CD in patients with psoriasis. Methods Serum antigliadin antibody IgG and IgA, antiendomysial antibody IgA and anti-transglutaminase antibody IgA were studied in 37 (19 males) patients with psoriasis and 50 (23 males) healthy controls. Upper gastrointestinal endoscopy and duodenal biopsies were performed in patients with at least one positive marker. Results Antigliadin IgA was statistically higher in the psoriasis group than in the controls (p<0.05). Serological markers were found positive in 6 patients with psoriasis and 1 person from the control group. Upper gastrointestinal endoscopy was performed in all these persons, with biopsies collected from the duodenum. The diagnosis of CD was reported in only one patient with psoriasis following the pathological examination of the biopsies. Whereas one person of the control group was found to be positive for antigliadin antibody IgA, pathological examination of the duodenal biopsies obtain from this patient were found to be normal. Conclusion Antigliadin IgA prominently increases in patients diagnosed with psoriasis. Patients with psoriasis should be investigated for latent CD and should be followed up. PMID:24003271

  3. Antiphospholipid antibodies: Paradigm in transition

    PubMed Central

    Horstman, Lawrence L; Jy, Wenche; Bidot, Carlos J; Ahn, Yeon S; Kelley, Roger E; Zivadinov, Robert; Maghzi, Amir H; Etemadifar, Masoud; Mousavi, Seyed Ali; Minagar, Alireza

    2009-01-01

    Objectives This is a critical review of anti-phospholipid antibodies (aPL). Most prior reviews focus on the aPL syndrome (APS), a thrombotic condition often marked by neurological disturbance. We bring to attention recent evidence that aPL may be equally relevant to non-thrombotic autoimmune conditions, notably, multiple sclerosis and ITP. Organization After a brief history, the recent proliferation of aPL target antigens is reviewed. The implication is that many more exist. Theories of aPL in thrombosis are then reviewed, concluding that all have merit but that aPL may have more diverse pathological consequences than now recognized. Next, conflicting results are explained by methodological differences. The lupus anticoagulant (LA) is then discussed. LA is the best predictor of thrombosis, but why this is true is not settled. Finally, aPL in non-thrombotic disorders is reviewed. Conclusion The current paradigm of aPL holds that they are important in thrombosis, but they may have much wider clinical significance, possibly of special interest in neurology. PMID:19154576

  4. Antibodies to watch in 2010

    PubMed Central

    2010-01-01

    Monoclonal antibodies (mAbs) are a burgeoning class of therapeutics, with more than 25 approved in countries worldwide. Novel molecules are entering clinical study at a rate of nearly 40 per year, and the commercial pipeline includes approximately 240 mAb therapeutics in clinical studies that have not yet progressed to regulatory approval or been approved. Of particular interest are the 26 mAbs that are currently at Phase 3, when safety and efficacy data critical to approval is established. Phase 3 study lengths are typically two to four years, so results for some studies might be announced in 2010, but data from others might not be presented until 2014. This overview of the 26 candidates provides a brief description of the background and the on-going Phase 3 studies of each mAb. Additional mAbs that have progressed to regulatory review or been approved may also be in Phase 3 studies, but these, as well as Fc fusion proteins, have been excluded. Due to the large body of primary literature about the 26 candidates, only selected references are given, with a focus on recent publications and articles that were relevant to Phase 3 studies. Current as of October 2009, the results presented here will serve as a baseline against which future progress can be measured. PMID:20065640

  5. HIV-1 resistance to neutralizing antibodies: Determination of antibody concentrations leading to escape mutant evolution.

    PubMed

    Magnus, Carsten; Reh, Lucia; Trkola, Alexandra

    2016-06-15

    Broadly neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) are considered vital components of novel therapeutics and blueprints for vaccine research. Yet escape to even the most potent of these antibodies is imminent in natural infection. Measures to define antibody efficacy and prevent mutant selection are thus urgently needed. Here, we derive a mathematical framework to predict the concentration ranges for which antibody escape variants can outcompete their viral ancestors, referred to as mutant selection window (MSW). When determining the MSW, we focus on the differential efficacy of neutralizing antibodies against HIV-1 in two canonical infection routes, free-virus infection and cell-cell transmission. The latter has proven highly effective in vitro suggesting its importance for both in vivo spread as well as for escaping targeted intervention strategies. We observed a range of MSW patterns that highlight the potential of mutants to arise in both transmission pathways and over wide concentration ranges. Most importantly, we found that only when the arising mutant has both, residual sensitivity to the neutralizing antibody and reduced infectivity compared to the parental virus, antibody dosing outside of the MSW to restrict mutant selection is possible. Emergence of mutants that provide complete escape and have no considerable fitness loss cannot be prevented by adjusting antibody doses. The latter may in part explain the ubiquitous resistance to neutralizing antibodies observed in natural infection and antibody treatment. Based on our findings, combinations of antibodies targeting different epitopes should be favored for antibody-based interventions as this may render complete resistance less likely to occur and also increase chances that multiple escapes result in severe fitness loss of the virus making longer-term antibody treatment more feasible. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  6. Immune complexes with cationic antibodies deposit in glomeruli more effectively than cationic antibodies alone.

    PubMed

    Mannik, M; Gauthier, V J; Stapleton, S A; Agodoa, L Y

    1987-06-15

    In previously published studies, highly cationized antibodies alone and in immune complexes bound to glomeruli by charge-charge interaction, but only immune complexes persisted in glomeruli. Because normal IgG does not deposit in glomeruli, studies were conducted to determine whether cationized antibodies can be prepared which deposit in glomeruli when bound to antigen but not when free in circulation. A series of cationized rabbit antiHSA was prepared with the number of added amino groups ranging from 13.3 to 60.2 per antibody molecule. Antibodies alone or in preformed soluble immune complexes, prepared at fivefold or 50-fold antigen excess, were administered to mice. With the injection of a fixed dose of 100 micrograms per mouse, antibodies alone did not deposit in glomeruli with less than 29.6 added amino groups by immunofluorescence microscopy. In contrast, 100 micrograms of antibodies with 23.5 added amino groups in immune complexes, made at fivefold antigen excess, formed immune deposits in glomeruli. With selected preparations of cationized, radiolabeled antibodies, deposition in glomeruli was quantified by isolation of mouse glomeruli. These quantitative data were in good agreement with the results of immunofluorescence microscopy. Immune complexes made at 50-fold antigen excess, containing only small-latticed immune complexes with no more than two antibody molecules per complex, deposited in glomeruli similar to antibodies alone. Selected cationized antibodies alone or in immune complexes were administered to mice in varying doses. In these experiments, glomerular deposition of immune complexes, made at fivefold antigen excess, was detected with five- to 10-fold smaller doses than the deposition of the same antibodies alone. These studies demonstrate that antibody molecules in immune complexes are more likely to deposit in glomeruli by charge-charge interactions than antibodies alone.

  7. Molecular stabilization effects of interactions between anti-metatype antibodies and liganded antibody.

    PubMed

    Weidner, K M; Denzin, L K; Voss, E W

    1992-05-25

    Anti-metatype antibodies have been described as antibodies which recognize ligand-induced conformational changes in the antibody variable region. Additionally, anti-metatype antibodies, produced by multiple immunizations with liganded high affinity monoclonal anti-fluorescein antibody 4-4-20, enhanced the lifetime of monoclonal antibody 4-4-20-fluorescein complex. To better understand the mechanism of the delayed dissociation rate, deuterium oxide was used to probe the liganded active site. The rate and extent of deuterium oxide-mediated fluorescence enhancement of bound ligand served to monitor the conformational dynamics of the active site in the presence and absence of anti-metatype antibodies. Results showed that anti-metatype antibodies reduced the rate and extent of deuterium oxide-mediated fluorescence enhancement of 4-4-20, a single-chain derivative of 4-4-20 (consisting of the variable domains and a polylinker), and idiotypically related monoclonal anti-fluorescein antibodies suggesting that anti-metatype stabilized the liganded active site. Size exclusion liquid chromatography was utilized to isolate the liganded antibody-anti-metatype complex. Liganded single chain antibody 4-4-20 was mixed with 10-fold molar excess anti-metatype Fab fragments, and a major complex eluted with an apparent M(r) 249,000. The apparent molecular weight of this complex inferred that one liganded single chain antibody was bound by five antimetatype Fab fragments. Spectral analysis confirmed these results and the characteristic delayed rate of ligand dissociation was also observed for the isolated complex. The results suggest that anti-metatype antibodies stabilize the liganded conformation by forming a large, stable, macromolecular complex.

  8. Significance of antibody orientation unraveled: well-oriented antibodies recorded high binding affinity.

    PubMed

    Tajima, Nobuyuki; Takai, Madoka; Ishihara, Kazuhiko

    2011-03-15

    To investigate the effect of antibody orientation on its immunological activities, we developed a novel and versatile platform consisting of a well-defined phospholipid polymer surface on which staphylococcal protein A (SpA) was site-selectively immobilized. The application of a biocompatible phospholipid-based platform ensured minimal denaturation of immobilized antibodies, and the site-selective immobilization of SpA clarified the effect of antibody orientation on immunological activities. The phospholipid polymer platform was prepared on silicon substrates using the surface-initiated atom transfer radical polymerization (SI-ATRP) technique. An enzymatic reaction was performed for orientation-selective coupling of SpA molecules to the polymer brush surface. Orientation-controlled antibodies were achieved using enzymatic reactions, and these antibodies captured 1.8 ± 0.1 antigens on average, implying that at least 80% of immobilized antibodies reacted with two antigens. Theoretical multivalent binding analysis further revealed that orientation-controlled antibodies had antigen-antibody reaction equilibrium dissociation constants (K(d)) as low as 8.6 × 10(-10) mol/L, whereas randomly oriented and partially oriented antibodies showed K(d) values of 2.0 × 10(-7) and 1.2 × 10(-7) mol/L, respectively. Strict control of antibody orientation not only formed an approximately 100-fold stronger antigen-antibody complex than the controls but also sustained the native antibody K(d) (10(-10)-10(-9) mol/L). These findings support the significance of antibody orientation because controlling the orientation resulted in high reactivity and theoretical binding capacity.

  9. Antibodies to Shiga toxins in Brazilian cattle.

    PubMed

    Yamamoto, Bruno B; Luz, Daniela; Abreu, Patrícia A E; Gotti, Tatiana B; Vasconcellos, Silvio A; Piazza, Roxane M F; Horton, Denise S P Q

    2017-07-01

    Cattle are considered a reservoir of Shiga toxin-producing Escherichia coli (STEC). There is no information about the presence of antibodies against Shiga toxins in Brazilian bovine serum. Using ELISA, all sera tested showed antibodies against the two main STEC virulence factors; Stx1 and Stx2. Neutralizing antibodies against Stx1 and/or Stx2 were detected in all but one serum. In conclusion, our results indicated that these animals had been exposed to STEC producing both toxins. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Intellectual property protection: strategies for antibody inventions.

    PubMed

    Storz, Ulrich

    2011-01-01

    In the last decade, therapeutic antibodies have become one of the commercially most successful classes of biopharmaceutical drugs. Major drug manufacturers who have successfully managed to occupy this new market, as well as biotechnology firms, some of which have experienced a quick growth and are now on par with the former, owe part of their success to suitable intellectual property strategies. This article provides an overview of the current thinking on antibody-related patents, and discusses strategies for protecting the antibody products of the future.

  11. Reshaping Human Antibodies: Grafting an Antilysozyme Activity

    NASA Astrophysics Data System (ADS)

    Verhoeyen, Martine; Milstein, Cesar; Winter, Greg

    1988-03-01

    The production of therapeutic human monoclonal antibodies by hybridoma technology has proved difficult, and this has prompted the ``humanizing'' of mouse monoclonal antibodies by recombinant DNA techniques. It was shown previously that the binding site for a small hapten could be grafted from the heavy-chain variable domain of a mouse antibody to that of a human myeloma protein by transplanting the hypervariable loops. It is now shown that a large binding site for a protein antigen (lysozyme) can also be transplanted from mouse to human heavy chain. The success of such constructions may be facilitated by an induced-fit mechanism.

  12. Immunocytochemical and Immunohistochemical Staining with Peptide Antibodies.

    PubMed

    Friis, Tina; Pedersen, Klaus Boberg; Hougaard, David; Houen, Gunnar

    2015-01-01

    Peptide antibodies are particularly useful for immunocytochemistry (ICC) and immunohistochemistry (IHC), where antigens may denature due to fixation of tissues and cells. Peptide antibodies can be made to any defined sequence, including unknown putative proteins and posttranslationally modified sequences. Moreover, the availability of large amounts of the antigen (peptide) allows inhibition/adsorption controls, which are important in ICC/IHC, due to the many possibilities for false-positive reactions caused by immunoglobulin Fc receptors, nonspecific reactions, and cross-reactivity of primary and secondary antibodies with other antigens and endogenous immunoglobulins, respectively. Here, simple protocols for ICC and IHC are described together with recommendations for appropriate controls.

  13. Solid-phase biotinylation of antibodies.

    PubMed

    Strachan, Elizabeth; Mallia, A Krishna; Cox, Joanna M; Antharavally, Babu; Desai, Surbhi; Sykaluk, Laura; O'Sullivan, Valerie; Bell, Peter A

    2004-01-01

    Biotinylation is an established method of labeling antibody molecules for several applications in life science research. Antibody functional groups such as amines, cis hydroxyls in carbohydrates or sulfhydryls may be modified with a variety of biotinylation reagents. Solution-based biotinylation is accomplished by incubating antibody in an appropriate buffered solution with biotinylation reagent. Unreacted biotinylation reagent must be removed via dialysis, diafiltration or desalting. Disadvantages of the solution-based approach include dilution and loss of antibody during post-reaction purification steps, and difficulty in biotinylation and recovery of small amounts of antibody. Solid-phase antibody biotinylation exploits the affinity of mammalian IgG-class antibodies for nickel IMAC (immobilized metal affinity chromatography) supports. In this method, antibody is immobilized on a nickel-chelated chromatography support and derivitized on-column. Excess reagents are easily washed away following reaction, and biotinylated IgG molecule is recovered under mild elution conditions. Successful solid phase labeling of antibodies through both amine and sulfhydryl groups is reported, in both column and mini-spin column formats. Human or goat IgG was bound to a Ni-IDA support. For sulfhydryl labeling, native disulfide bonds were reduced with TCEP, and reduced IgG was biotinylated with maleimide-PEO(2) biotin. For amine labeling, immobilized human IgG was incubated with a solution of NHS-PEO(4) biotin. Biotinylated IgG was eluted from the columns using a buffered 0.2 M imidazole solution and characterized by ELISA, HABA/avidin assay, probing with a streptavidin-alkaline phosphatase conjugate, and binding to a monomeric avidin column. The solid phase protocol for sulfhydryl labeling is significantly shorter than the corresponding solution phase method. Biotinylation in solid phase is convenient, efficient and easily applicable to small amounts of antibody (e.g. 100 microg

  14. Serum Antibodies to Glycans in Peripheral Neuropathies.

    PubMed

    Sonnino, Sandro; Chiricozzi, Elena; Ciampa, Maria Grazia; Mauri, Laura; Prinetti, Alessandro; Toffano, Gino; Aureli, Massimo

    2017-03-01

    In peripheral neuropathies, such as sensorimotor neuropathies, motor neuron diseases, or the Guillain-Barré syndrome, serum antibodies recognizing saccharide units, portion of oligosaccharides, or oligosaccharide chains, have been found. These antibodies are called anti-glycosphingolipid (GSL) or anti-ganglioside antibodies. However, the information on the aglycone carrying the hydrophilic oligosaccharide remains elusive. The absolute and unique association of GSL to the onset, development and symptomatology of the peripheral neuropathies could be misleading. Here, we report some thoughts on the matter.

  15. Uses of monoclonial antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V.

    2015-06-23

    This invention provides an antibody that binds the same antigen as that of monoclonal antibody 8H9, wherein the heavy chain CDR (Complementary Determining Region)1 comprises NYDIN, heavy chain CDR2 comprises WIFPGDGSTQY, heavy chain CDR3 comprises QTTATWFAY, and the light chain CDR1 comprises RASQSISDYLH, light chain CDR2 comprises YASQSIS, and light chain CDR3 comprises QNGHSFPLT. In another embodiment, there is provided a polypeptide that binds the same antigen as that of monoclonal antibody 8H9, wherein the polypeptide comprises NYDIN, WIFPGDGSTQY, QTTATWFAY, RASQSISDYLH, YASQSIS, and QNGHSFPLT.

  16. The antibody approach of labeling blood cells

    SciTech Connect

    Srivastava, S.C.

    1992-12-31

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are critically assessed and evaluated.

  17. The antibody approach of labeling blood cells

    SciTech Connect

    Srivastava, S.C.

    1991-12-31

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

  18. The antibody approach of labeling blood cells

    SciTech Connect

    Srivastava, S.C.

    1991-01-01

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

  19. Complement-fixing antibodies against denatured HLA and MICA antigens are associated with antibody mediated rejection.

    PubMed

    Cai, Junchao; Terasaki, Paul I; Zhu, Dong; Lachmann, Nils; Schönemann, Constanze; Everly, Matthew J; Qing, Xin

    2016-02-01

    We have found antibodies against denatured HLA class I antigens in the serum of allograft recipients which were not significantly associated with graft failure. It is unknown whether transplant recipients also have denatured HLA class II and MICA antibodies. The effects of denatured HLA class I, class II, and MICA antibodies on long-term graft outcome were further investigated based on their ability to fix complement c1q. In this 4-year retrospective cohort study, post-transplant sera from 975 kidney transplant recipients were tested for antibodies against denatured HLA/MICA antigens and these antibodies were further classified based on their ability to fix c1q. Thirty percent of patients had antibodies against denatured HLA class I, II, or MICA antigens. Among them, 8.5% and 21.5% of all patients had c1q-fixing and non c1q-fixing antibodies respectively. There was no significant difference on graft survival between patients with or without antibodies against denatured HLA/MICA. However, when these antibodies were further classified according to their ability to fix c1q, patients with c1q-fixing antibodies had a significantly lower graft survival rate than patients without antibodies or patients with non c1q-fixing antibodies (p=0.008). In 169 patients who lost renal grafts, 44% of them had c1q-fixing antibodies against denatured HLA/MICA antigens, which was significantly higher than that in patients with functioning renal transplants (25%, p<0.0001). C1q-fixing antibodies were more significantly associated with graft failure caused by AMR (72.73%) or mixed AMR/CMR (61.9%) as compared to failure due to CMR (35.3%) or other causes (39.2%) (p=0.026). Transplant recipients had antibodies against denatured HLA class I, II, and MICA antigens. However, only c1q-fixing antibodies were associated with graft failure which was related to antibody mediated rejection. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Fc engineering of antibodies and antibody derivatives by primary sequence alteration and their functional characterization.

    PubMed

    Derer, Stefanie; Kellner, Christian; Rösner, Thies; Klausz, Katja; Glorius, Pia; Valerius, Thomas; Peipp, Matthias

    2014-01-01

    Therapeutic antibodies used in the treatment of cancer patients are able to mediate diverse effector mechanisms. Dependent on tumor entity, localization, and tumor burden different effector mechanisms may contribute to the in vivo antitumor activity to a variable degree. Especially Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) have been suggested as being important for the in vivo activity of therapeutic antibodies like rituximab or trastuzumab. In recent years, several strategies have been pursued to further optimize the cytotoxic potential of monoclonal antibodies by modifying their Fc part (Fc engineering) with the ultimate goal to enhance antibody therapy.Since Fc engineering approaches are applicable to any Fc-containing molecule, strategies to enhance CDC or ADCC activity of full antibodies or scFv-Fc fusion proteins by altering the primary Fc sequence are described.

  1. Advancements in nanosensors using plastic antibodies.

    PubMed

    Volkert, Anna A; Haes, Amanda J

    2014-01-07

    Biosensors possess recognition elements that bind to target molecules which lead to detectable signals. Incorporation of noble metal nanomaterials into biosensors allows for rapid and simple biomolecule detection. Herein, recent developments in affinity nanosensors will be discussed. These sensors often include naturally occurring recognition elements such as antibodies and DNA. As samples become more complex, new recognition elements are sought. For instance, plastic antibodies provide alternative and more environmentally stable recognition elements than traditional antibodies. Molecular imprinted polymers, a class of plastic antibodies, promote biomolecule recognition and detection. The incorporation of noble metal nanomaterials into molecular imprinted polymer biosensors for real world applications will be explored. Further improvements in the design of artificial recognition agents are envisioned to facilitate new methods for complex biological and chemical analyses.

  2. [Antibody formation in type A viral hepatitis].

    PubMed

    Teokharova, M P; Losev, B B; Andonov, A P; Draganov, P I; Mineva, E G

    1978-01-01

    Immune response to hepatitis type A antigen (HAAg) was measured by the passive hemagglutination test (PHA) and by the immune adherence test (IAHA). Specific antibodies found by PHA are of the IgM class which indicates a recent exposure to hepatitis A virus. The antibodies found by IAHA reflect the level of postinfectious immune status. The antibody curve is highest in the age group of 30--49 years (95%). The above-mentioned serological tests were carried out with purified by gel filtration in Sepharose 6B Botevgrad faecal morphologically consisting of 27 nm particles with the buoyant density in CcCl of 1.40 g/ml. The same particles were aggregated with sera positive for antibody to hepatitis type A antigen in immune electron microscopy (IEM).

  3. Solid phase synthesis of bifunctional antibodies.

    PubMed

    DeSilva, B S; Wilson, G S

    1995-12-15

    Bifunctional antibodies were prepared using the principle of solid-phase synthesis. The two Fab' fragments were chemically linked together via a bismaleimide crosslinking reagent. The F(ab')2 fragments from intact IgG were prepared using an immobilized pepsin column. Goat, mouse and human antibodies were digested completely within 4 h. The F(ab')2 fragments thus produced did not contain any IgG impurities. The Fab' fragments were produced by reducing the inter-heavy chain disulfide bonds using 2-mercaptoethylamine. The use of the solid-phase reactor in the preparation of the bifunctional antibodies eliminated many of the time-consuming separation steps between the fragmentation and conjugation steps. This procedure facilitates the automation of the bifunctional antibody preparation and the rapid optimization of reaction conditions.

  4. Synthesis of bifunctional antibodies for immunoassays.

    PubMed

    DeSilva, B S; Wilson, G S

    2000-09-01

    The synthesis of bifunctional antibodies using the principle of solid-phase synthesis is described. Two Fab' fragments were chemically linked together via a bismaleimide crosslinking reagent. The F(ab')(2) fragments from intact immunoglobulin G (IgG) were prepared using an immobilized pepsin column. Goat, mouse, and human antibodies were digested completely within 4 h. The F(ab')(2) fragments thus produced did not contain any IgG impurities. Fab' fragments were produced by reducing the heavy interchain disulfide bonds using 2-mercaptoethylamine. Use of the solid-phase reactor in the preparation of the bifunctional antibodies eliminated many of the time-consuming separation steps between the fragmentation and conjugation steps. This procedure facilitates the automation of bifunctional antibody preparation and the rapid optimization of reaction conditions.

  5. Patient-Derived Antibody Targets Tumor Cells

    Cancer.gov

    An NCI Cancer Currents blog on an antibody derived from patients that killed tumor cells in cell lines of several cancer types and slowed tumor growth in mouse models of brain and lung cancer without evidence of side effects.

  6. Monoclonal antibodies against metallothioneins and metalloproteins.

    PubMed

    Talbot, B G; Bilodeau, G; Thirion, J P

    1986-10-01

    Monoclonal antibodies against rabbit metallothioneins (MT) were prepared by in vitro immunization of mouse lymphocytes with a mixture of the two forms of metallothionein MT1 and MT2. Six IgM antibodies (TN1,3,4,5,6,7) which bind to metallothionein were characterized. Antibody TN3 is specific for rabbit MT1 and does not react with any other MT's tested. TN5 is specific for both rabbit MT1 and MT2. TN7 is specific for rabbit MT2 but not MT1 and cross-reacts also with Chinese hamster, mouse and rat metallothioneins. The antibodies TN1, TN4 and TN6 bind not only to rabbit MT1 and MT2 but also to other metal binding proteins like alcohol dehydrogenase and carbonic anhydrase.

  7. Antinuclear antibodies specific for primary biliary cirrhosis.

    PubMed

    Worman, Howard J; Courvalin, Jean Claude

    2003-06-01

    The serological hallmark of primary biliary cirrhosis (PBC) is the presence of antimitochondrial antibodies. However, antinuclear antibodies (ANA) are also detectable in approximately 50% of subjects with PBC. Most clinical laboratories use indirect immunofluorescence microscopy to detect ANA and two labeling patterns that predominate in PBC are 'punctate nuclear rim' and 'multiple nuclear dots.' Work over the past several years has shown that antibodies giving these patterns most often recognize nuclear pore membrane protein gp210 and nuclear body protein sp100, respectively. These ANA are highly specific for PBC and detected in approximately 25% of patients. Less frequently, ANA apparently unique to PBC recognize other proteins of the nuclear envelope and nuclear bodies. While antibodies against gp210, sp100 and some other nuclear proteins are very specific to PBC and may therefore be useful diagnostic markers, their connection to pathogenesis remains to be elucidated.

  8. Antiphospholipid antibodies in critically ill patients

    PubMed Central

    Vassalo, Juliana; Spector, Nelson; de Meis, Ernesto; Soares, Márcio; Salluh, Jorge Ibrain Figueira

    2014-01-01

    Antiphospholipid antibodies are responsible for a wide spectrum of clinical manifestations. Venous, arterial and microvascular thrombosis and severe catastrophic cases account for a large morbidly/mortality. Through the connection between the immune, inflammatory and hemostatic systems, it is possible that these antibodies may contribute to the development of organ dysfunction and are associated with poor short and long-term prognoses in critically ill patients. We performed a search of the PubMed/MedLine database for articles written during the period from January 2000 to February 2013 to evaluate the frequency of antiphospholipid antibodies in critically ill patients and their impact on the outcomes of these patients. Only eight original studies involving critically ill patients were found. However, the development of antiphospholipid antibodies in critically ill patients seems to be frequent, but more studies are necessary to clarify their pathogenic role and implications for clinical practice. PMID:25028953

  9. Gut Microbial Metabolites Fuel Host Antibody Responses.

    PubMed

    Kim, Myunghoo; Qie, Yaqing; Park, Jeongho; Kim, Chang H

    2016-08-10

    Antibody production is a metabolically demanding process that is regulated by gut microbiota, but the microbial products supporting B cell responses remain incompletely identified. We report that short-chain fatty acids (SCFAs), produced by gut microbiota as fermentation products of dietary fiber, support host antibody responses. In B cells, SCFAs increase acetyl-CoA and regulate metabolic sensors to increase oxidative phosphorylation, glycolysis, and fatty acid synthesis, which produce energy and building blocks supporting antibody production. In parallel, SCFAs control gene expression to express molecules necessary for plasma B cell differentiation. Mice with low SCFA production due to reduced dietary fiber consumption or microbial insufficiency are defective in homeostatic and pathogen-specific antibody responses, resulting in greater pathogen susceptibility. However, SCFA or dietary fiber intake restores this immune deficiency. This B cell-helping function of SCFAs is detected from the intestines to systemic tissues and conserved among mouse and human B cells, highlighting its importance.

  10. Recurrent adverse pregnancy outcome and antiphospholipid antibodies.

    PubMed

    Reece, E A; Gabrielli, S; Cullen, M T; Zheng, X Z; Hobbins, J C; Harris, E N

    1990-07-01

    Antiphospholipid antibodies, which include lupus-like anticoagulant and anticardiolipin antibody, have been linked to a number of adverse pregnancy outcomes, although their exact pathogenic mechanisms remain poorly defined. The relative risk of complications such as intrauterine growth retardation, spontaneous abortions, and stillbirth in patients with antiphospholipid antibodies also remains undetermined. Heightened attention has been focused on the association, leading to investigations into the pathogenesis. Uncontrolled studies have also explored therapeutic regimens such as aspirin, steroids, and heparin, and clinical trials have used various treatment protocols. Although knowledge into the association of antiphospholipid antibodies and recurrent adverse pregnancy outcome is limited and continues to evolve, this association provides new insights into the disease and offers promise for pharmacologic prophylaxis. In this article, current concepts on pathogenesis, diagnosis, and therapy are reviewed and recommendations are made for clinical care of these patients.

  11. Creation of catalytic antibodies metabolizing organophosphate compounds.

    PubMed

    Kurkova, I N; Smirnov, I V; Belogurov, A A; Ponomarenko, N A; Gabibov, A G

    2012-10-01

    Development of new ways of creating catalytic antibodies possessing defined substrate specificity towards artificial substrates has important fundamental and practical aspects. Low immunogenicity combined with high stability of immunoglobulins in the blood stream makes abzymes potent remedies. A good example is the cocaine-hydrolyzing antibody that has successfully passed clinical trials. Creation of an effective antidote against organophosphate compounds, which are very toxic substances, is a very realistic goal. The most promising antidotes are based on cholinesterases. These antidotes are now expensive, and their production methods are inefficient. Recombinant antibodies are widely applied in clinics and have some advantage compared to enzymatic drugs. A new potential abzyme antidote will combine effective catalysis comparable to enzymes with high stability and the ability to switch on effector mechanisms specific for antibodies. Examples of abzymes metabolizing organophosphate substrates are discussed in this review.

  12. Brain-Reactive Antibodies and Disease

    PubMed Central

    Diamond, B.; Honig, G.; Mader, S.; Brimberg, L.; Volpe, B.T.

    2015-01-01

    Autoimmune diseases currently affect 5–7% of the world's population; in most diseases there are circulating autoantibodies. Brain-reactive antibodies are present in approximately 2–3% of the general population but do not usually contribute to brain pathology. These antibodies penetrate brain tissue only early in development or under pathologic conditions. This restriction on their pathogenicity and the lack of correlation between serum titers and brain pathology have, no doubt, contributed to a delayed appreciation of the contribution of autoantibodies in diseases of the central nervous system. Nonetheless, it is increasingly clear that antibodies can cause damage in the brain and likely initiate or aggravate multiple neurologic conditions; brain-reactive antibodies contribute to symptomatology in autoimmune disease, infectious disease, and malignancy. PMID:23516983

  13. Acute myeloid leukemia targets for bispecific antibodies

    PubMed Central

    Hoseini, S S; Cheung, N K

    2017-01-01

    Despite substantial gains in our understanding of the genomics of acute myelogenous leukemia (AML), patient survival remains unsatisfactory especially among the older age group. T cell-based therapy of lymphoblastic leukemia is rapidly advancing; however, its application in AML is still lagging behind. Bispecific antibodies can redirect polyclonal effector cells to engage chosen targets on leukemia blasts. When the effector cells are natural-killer cells, both antibody-dependent and antibody-independent mechanisms could be exploited. When the effectors are T cells, direct tumor cytotoxicity can be engaged followed by a potential vaccination effect. In this review, we summarize the AML-associated tumor targets and the bispecific antibodies that have been studied. The potentials and limitations of each of these systems will be discussed. PMID:28157217

  14. Broadly neutralizing antibodies against influenza viruses

    PubMed Central

    Laursen, Nick S.; Wilson, Ian A.

    2014-01-01

    Despite available antivirals and vaccines, influenza infections continue to be a major cause of mortality worldwide. Vaccination generally induces an effective, but strain-specific antibody response. As the virus continually evolves, new vaccines have to be administered almost annually when a novel strain becomes dominant. Furthermore, the sporadic emerging resistance to neuraminidase inhibitors among circulating strains suggests an urgent need for new therapeutic agents. Recently, several cross-reactive antibodies have been described, which neutralize an unprecedented spectrum of influenza viruses. These broadly neutralizing antibodies generally target conserved functional regions on the major influenza surface glycoprotein hemagglutinin (HA). The characterization of their neutralization breadth and epitopes on HA could stimulate the development of new antibody-based antivirals and broader influenza vaccines. PMID:23583287

  15. Polynucleotides encoding anti-sulfotyrosine antibodies

    DOEpatents

    Bertozzi, Carolyn R [Berkeley, CA; Kehoe, John [Saint Davids, PA; Bradbury, Andrew M [Santa Fe, NM

    2011-01-11

    The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

  16. Antibodies as Mediators of Brain Pathology

    PubMed Central

    Brimberg, Lior; Mader, Simone; Fujieda, Yuichiro; Arinuma, Yoshiyuki; Kowal, Czeslawa; Volpe, Bruce T.; Diamond, Betty

    2016-01-01

    The brain is normally sequestered from antibody exposure by the blood brain barrier. However, antibodies can access the brain during fetal development before the barrier achieves full integrity, and in disease states when barrier integrity is compromised. Recent studies suggest that antibodies contribute to brain pathology associated with autoimmune diseases such as systemic lupus erythematosus and neuromyelitis optica, and can lead to transient or permanent behavioral or cognitive abnormalities. We review these findings here and examine the circumstances associated with antibody entry into the brain, the routes of access and the mechanisms that then effect pathology. Understanding these processes and the nature and specificity of neuronal autoantibodies may reveal therapeutic strategies toward alleviating or preventing the neurological pathologies and behavioral abnormalities associated with autoimmune disease. PMID:26494046

  17. Deep sequencing and human antibody repertoire analysis

    PubMed Central

    Boyd, Scott D; Crowe, James E

    2016-01-01

    In the past decade, high-throughput DNA sequencing (HTS) methods and improved approaches for isolating antigen-specific B cells and their antibody genes have been applied in many areas of human immunology. This work has greatly increased our understanding of human antibody repertoires and the specific clones responsible for protective immunity or immune-mediated pathogenesis. Although the principles underlying selection of individual B cell clones in the intact immune system are still under investigation, the combination of more powerful genetic tracking of antibody lineage development and functional testing of the encoded proteins promises to transform therapeutic antibody discovery and optimization. Here, we highlight recent advances in this fast-moving field. PMID:27065089

  18. Enzyme mimicry by the antiidiotypic antibody approach

    PubMed Central

    Kolesnikov, Alexander V.; Kozyr, Arina V.; Alexandrova, Elena S.; Koralewski, Frédéric; Demin, Alexander V.; Titov, Mikhail I.; Avalle, Bérangère; Tramontano, Alfonso; Paul, Sudhir; Thomas, Daniel; Gabibov, Alexander G.; Friboulet, Alain

    2000-01-01

    The concept of “internal image” of antiidiotypic antibodies has provided the basis for eliciting catalytic antibodies. A monoclonal IgM 9A8 that was obtained as an antiidiotype to AE-2 mAb, a known inhibitor of acetylcholinesterase, displayed esterolytic activity. Study of recombinant Fab fragments and separate light and heavy chains of 9A8 confirmed that the antibody variable domain encodes the catalytic function, whereas neither part of the primary sequence of the Fab exhibited homology with the enzyme. The specific modification of the 9A8 variable domain by an active site-directed covalent inhibitor revealed the presence of an active site Ser residue. A three-dimensional modeling suggests the existence of a functional catalytic dyad Ser-His. Comparison of active sites of 9A8 and 17E8 esterolytic abzyme raised against transition-state analog revealed structural similarity although both antibodies were elicited by two different approaches. PMID:11095704

  19. Antiphospholipid antibodies investigation in Pneumocystis jirovecii carriers.

    PubMed

    Atienza, Maria R; Respaldiza, Nieves; De La Santa, Eva; Martín-Garrido, Isabel; Medrano, Francisco J; Varela, José M; Calderón, Enrique

    2008-01-01

    It is well documented that antiphospholipid antibodies are increased in patients with HIV-1 infection and these are most commonly seen in those with Pneumocystis jirovecii pneumonia. Therefore it has been proposed that this could be the cause of its presence. Recently, P. jirovecii subclinical infection has been described in non-immunodeficient patients. We report here our experience concerning the possible relationship between P. jirovecii infection in non-immunocompromized adults and the production of antiphospholipid antibodies. Circulating lupus anticoagulant and IgM anticardiolipin antibodies were negative in all patients. IgG anticardiolipin antibodies were positive in 2 out of 5 (40%) P. jirovecii carriers and 2 out of 10 (20%) subjects with no evidence of pulmonary infection by this microorganism (p=0.4).

  20. Monoclonal Antibodies for Multiple Sclerosis Treatment.

    PubMed

    Palavra, Filipe

    2015-01-01

    Since their introduction in medical therapy, in the last quarter of the 20th century, monoclonal antibodies have gained an increasing importance in the treatment of various diseases. Neurology has been one of the medical specialties benefiting of the therapeutic potential of these monoclonal antibodies and certain neurological conditions may now contain such drugs in their therapeutic algorithms. Multiple sclerosis is one of these diseases and, in addition to the monoclonal antibodies already licensed for clinical use, several others are in development for future utilization in this specific area. The future will certainly pass through this kind of drugs and, in this article, a review of the most relevant data related to monoclonal antibodies already in use and also in clinical development for multiple sclerosis treatment will be performed.

  1. Targeting Malignant Brain Tumors with Antibodies.

    PubMed

    Razpotnik, Rok; Novak, Neža; Čurin Šerbec, Vladka; Rajcevic, Uros

    2017-01-01

    Antibodies have been shown to be a potent therapeutic tool. However, their use for targeting brain diseases, including neurodegenerative diseases and brain cancers, has been limited, particularly because the blood-brain barrier (BBB) makes brain tissue hard to access by conventional antibody-targeting strategies. In this review, we summarize new antibody therapeutic approaches to target brain tumors, especially malignant gliomas, as well as their potential drawbacks. Many different brain delivery platforms for antibodies have been studied such as liposomes, nanoparticle-based systems, cell-penetrating peptides (CPPs), and cell-based approaches. We have already shown the successful delivery of single-chain fragment variable (scFv) with CPP as a linker between two variable domains in the brain. Antibodies normally face poor penetration through the BBB, with some variants sufficiently passing the barrier on their own. A "Trojan horse" method allows passage of biomolecules, such as antibodies, through the BBB by receptor-mediated transcytosis (RMT). Such examples of therapeutic antibodies are the bispecific antibodies where one binding specificity recognizes and binds a BBB receptor, enabling RMT and where a second binding specificity recognizes an antigen as a therapeutic target. On the other hand, cell-based systems such as stem cells (SCs) are a promising delivery system because of their tumor tropism and ability to cross the BBB. Genetically engineered SCs can be used in gene therapy, where they express anti-tumor drugs, including antibodies. Different types and sources of SCs have been studied for the delivery of therapeutics to the brain; both mesenchymal stem cells (MSCs) and neural stem cells (NSCs) show great potential. Following the success in treatment of leukemias and lymphomas, the adoptive T-cell therapies, especially the chimeric antigen receptor-T cells (CAR-Ts), are making their way into glioma treatment as another type of cell-based therapy using the

  2. Therapeutic monoclonal antibody for sporotrichosis

    PubMed Central

    Almeida, Sandro R.

    2012-01-01

    Sporotrichosis is a chronic subcutaneous mycosis that affects both humans and animals worldwide. This subcutaneous mycosis had been attributed to a single etiological agent, Sporothrix schenckii. S. schenckii exhibits considerable genetic variability, and recently, it was suggested that this taxon consists of a complex of species. Sporotrichosis is caused by traumatic inoculation of the fungus, which is a ubiquitous environmental saprophyte that can be isolated from soil and plant debris. The infection is limited to cutaneous forms, but recently, more severe clinical forms of this mycosis have been described, especially among immunocompromised individuals. The immunological mechanisms involved in the prevention and control of sporotrichosis are not well understood. Some studies suggest that cell-mediated immunity plays an important role in protecting the host against S. schenckii. In contrast, the role of the humoral immune response in protection against this fungus has not been studied in detail. In a previous study, we showed that antigens secreted by S. schenckii induced a specific humoral response in infected animals, primarily against a 70-kDa molecule, indicating a possible role of specific antibodies against this molecule in infection control. In another study by our group, we produced a mAb against a 70-kDa glycoprotein of S. schenckii to better understand the effect of the passive immunization of mice infected with S. schenckii. The results showed a significant reduction in the number of CFUs in various mice organs when the mAb was injected before or during S. schenckii infection. Similar results were observed when T-cell-deficient mice were used. The drugs of choice in the treatment of sporotrichosis require long periods, and relapses are frequently observed, primarily in immunocompromised patients. The strong protection induced by the mAb against a 70-kDa glycoprotein makes it a strong candidate as a therapeutic vaccine against sporotrichosis. PMID

  3. Function-first antibody discovery: Embracing the unpredictable biology of antibodies.

    PubMed

    Frendéus, Björn

    2013-08-01

    Therapeutic antibodies may mediate antineoplastic effects by altering the biological functions of their target, by directly stimulating the demise of cancer cells or by activating antibody-dependent immune effector mechanisms. We have recently provided in vivo proof-of-concept for a "function-first" target and drug discovery platform in which antibodies against a multitude of tumor-associated antigens are screened for biological effects in a target-unbiased manner.

  4. The current status of neutrophil cytoplasmic antibodies.

    PubMed Central

    van der Woude, F J; Daha, M R; van Es, L A

    1989-01-01

    Several studies in the past 10 years have demonstrated the occurrence of autoantibodies against cytoplasmic constituents in patients with vasculitis and glomerulonephritis. In this review the nomenclature of these antibodies is discussed and assays and clinical associations are summarized. Although the antigens involved are not completely identified, antibodies and T cells reactive with myeloid lysozomal enzymes may both play a significant role in pathogenesis. PMID:12412739

  5. Dengue Antibody and Zika: Friend or Foe?

    PubMed

    Durbin, Anna P

    2016-10-01

    Zika virus is a mosquito-borne Flavivirus related to dengue that is rapidly spreading through the Americas. This outbreak is occurring in dengue-endemic areas where the population has acquired antibodies to dengue. Recent studies reveal that preexisting dengue antibodies may have opposite effects on Zika infection, transmission, and clinical outcome. Discerning these effects is critical to a better understanding of Zika pathogenesis and the prevention of future outbreaks.

  6. History and Practice: Antibodies in Infectious Diseases.

    PubMed

    Hey, Adam

    2015-04-01

    Antibodies and passive antibody therapy in the treatment of infectious diseases is the story of a treatment concept which dates back more than 120 years, to the 1890s, when the use of serum from immunized animals provided the first effective treatment options against infections with Clostridium tetani and Corynebacterium diphtheriae. However, after the discovery of penicillin by Fleming in 1928, and the subsequent introduction of the much cheaper and safer antibiotics in the 1930s, serum therapy was largely abandoned. However, the broad and general use of antibiotics in human and veterinary medicine has resulted in the development of multi-resistant strains of bacteria with limited to no response to existing treatments and the need for alternative treatment options. The combined specificity and flexibility of antibody-based treatments makes them very valuable tools for designing specific antibody treatments to infectious agents. These attributes have already caused a revolution in new antibody-based treatments in oncology and inflammatory diseases, with many approved products. However, only one monoclonal antibody, palivizumab, for the prevention and treatment of respiratory syncytial virus, is approved for infectious diseases. The high cost of monoclonal antibody therapies, the need for parallel development of diagnostics, and the relatively small markets are major barriers for their development in the presence of cheap antibiotics. It is time to take a new and revised look into the future to find appropriate niches in infectious diseases where new antibody-based treatments or combinations with existing antibiotics, could prove their value and serve as stepping stones for broader acceptance of the potential for and value of these treatments.

  7. Primary Antiphospholipid Antibody Syndrome: A Case Report.

    PubMed

    Kadeli, Deepak K; Hanjagi, Siddaraya Y

    2015-10-01

    Primary Antiphospholipid antibody syndrome is a rare disease associated with thromboembolic events which may affect either the arterial or the venous vasculature. It presents with an increased risk of thrombosis in pregnant woman leading to repeated fetal losses. We present here a case of primary antiphospholipid antibody syndrome in young women who had previous event of gangrene of toes leading to their amputation and repeated fetal losses.

  8. Probing myosin head structure with monoclonal antibodies.

    PubMed

    Winkelmann, D A; Lowey, S

    1986-04-20

    Monoclonal antibodies that react with defined regions of the heavy and light chains of chicken skeletal muscle myosin have been used to provide a correlation between the primary and the tertiary structures of the head. Electron microscopy of rotary shadowed antibody-myosin complexes shows that the sites for three epitopes in the 25,000 Mr tryptic fragment (25k) of subfragment-1, including one within 4000 Mr of the amino terminus of the myosin heavy chain, are clustered 145(+/- 20) A from the head-rod junction. An epitope in the 50,000 Mr fragment maps even further out on the head. These antibodies bind to the head in several orientations, suggesting that each of the heads can rotate can rotate 180 degrees about the head-rod junction. The epitopes are accessible on subfragment-1 bound to actin when they were probed with Fab fragments; therefore, none of these heavy chain sites is is on the contact surface between the head and actin. Two of the anti-25k antibodies affect the K+-EDTA-and Ca2+-ATPase activities of myosin in a manner that mimics the effect on activity of the modification of the reactive thiol, SH-1. These two antibodies also inhibit the actin-activated ATPase non-competitively with respect to actin. None of the other eight antibodies tested had any marked effect on activity. A monoclonal antibody that reacts with an epitope in the amino-terminal third of myosin light chain 2 maps close to the head-rod junction. A polyclonal antibody specific for the amino terminus of light chain 3 binds further up in the "neck region" of the head, indicating that these portions of the two classes of light chains are located at different sites.

  9. Antibodies to Actin in Autoimmune Neutropenia

    DTIC Science & Technology

    1990-02-01

    protein as actin. Purified Acanthamoeba actin by anti-neutrophil antibodies in autoimmune neutropenia, comigrated with the protein and was specifically...anti-rabbit IgG were obtained from ICN Immunobiolog- formed using purified Acanthamoeba actin (gift of Dr Blair Bowers. icals, Naperville, IL. Cells...preparations𔃼 1 - was the protein recognized by these anti-neutrophil antibody 6 .2- positive sera, lgG, and F(ab’) 2. Purified Acanthamoeba actin

  10. Monoclonal antibodies as catalysts for cyanide removal

    SciTech Connect

    Cook, C.E.; Whisnant, C.C.; Miller, D.B.; Allen, D.A.; Basta, P.V.

    1993-05-13

    We have shown that hydrogen cyanide reacts with alpha, beta-unsaturated ketones to form stable compounds under physiological conditions (temperature, pH). Although spontaneous reaction is too slow for protection against cyanide intoxication, rate enhancement in the presence of a suitable catalyst would permit the use of alpha, beta-unsaturated ketones (enones) as prophylactics for cyanide exposure. Based on the accepted mechanism for this 1,4-addition reaction, we have designed and synthesized sized a transition state analog (TSA), conjugated it to protein and used the conjugate to produce more than 300 monoclonal antibodies which bind the TSA. Approximately 10% of these antibodies have been purified from ascites and tested for catalysis of the addition reaction of cyanide to enone. Product formation was measured by HPLC. Four antibodies have been found which significantly enhance the initial velocity of the reaction. The TSA markedly diminishes the reaction velocity, indicating the involvement of the antibody binding site in the observed enhancement. Preliminary kinetic analysis on one antibody gave values of K sub (enone) and K sub KCN 51 uM and 9.6 mM, respectively. The value of k sub (cat) was 2.33 hr-1. The data suggest a rate enhancement of 2 x 10 to the 4th power for the encounter of the enone with the antibody-cyanide complex, whereas the rate enhancement for encounter of cyanide with the antibody-enone complex is 70. To utilize the potential of genetic engineering for modifying the proper-lies of anti-TSA monoclonal antibodies, we are cloning heavy and light chain genes for sequencing and subsequent site-specific mutagenesis.

  11. The dilemma of DQ HLA-antibodies.

    PubMed

    Al Attas, Rabab; Al Abduladheem, Dalal; Shawhatti, Adel A; Lopez, Ricardo; Liacini, Abdelhamid; AlZahrani, Saber; Akkari, Khalid; Hasan, Nasreen; Alabadi, Abdulnaser

    2015-05-01

    Accurate identification of antibody reactivity against HLA-DQ antigens was difficult by using the old serological assays because of the strong linkage disequilibrium between HLA-DR and HLA-DQ (the usual inheritance of a certain HLA-DR molecule that ties together with the same DQ molecule within a racial group). The accurate and precise identifications of anti-HLA-antibodies of DQ specificities were made possible with the introduction of multiplex-bead arrays (Luminex), using single antigen bead (SAB) assay. The SAB assay is also considered today to be the most sensitive and specific method for alloimmunization assessment even for the low titer anti-HLA-antibodies. However, it is becoming clear that the detection of the HLA antibodies by SAB is not absolutely perfect due to the variation in densities, conformations and orientations of the antigen coated beads. Unlike HLA-DR, the HLA-DQ antigens are made of two polymorphic chains, both (alpha and beta chains) can contribute to the process of immunization individually or jointly. Routine SAB testing approach, which assigns the specificities based on beta chains and ignores the contribution of the DQα chains, can lead to erroneous DQ-antibody assignments. Therefore, it is important to recognize both the peculiarity of the HLA-DQ antigens as well as the nature of the assay format used in order to reach the correct antibody assignments. Erroneous donor specific antibodies (DSA) assignment may lead to denial of an otherwise immunologically compatible organ transplant, or exposing transplant recipients to unnecessary investigations or immunosuppression. The following two patients presented with HLA-antibodies against DQ antigens (anti-DQ-Abs) highlight these two scenarios. Copyright © 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  12. Antibody transport and internalization into tumours.

    PubMed Central

    Matzku, S.; Moldenhauer, G.; Kalthoff, H.; Canevari, S.; Colnaghi, M.; Schuhmacher, J.; Bihl, H.

    1990-01-01

    Internalization of monoclonal antibody (MAb) conjugates is an important feature of tumour targeting, both with respect to the therapeutic action of substances coupled to the antibody and to retention of radionuclides. Problems of analysing internalization in vitro and in vivo, of manipulating internalization, and of evaluating the involvement of normal tissues are illustrated by recent experimental data and are discussed in the light of published evidence. Images Figure 4 PMID:2383472

  13. Standardization of anti-DNA antibody assays.

    PubMed

    Pisetsky, David S

    2013-07-01

    Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus and represent important biomarkers for clinical and research purposes. These antibodies are part of a family of antibodies to nucleosomes and bind to conserved sites widely present on DNA. While the value of anti-DNA as a biomarker is well established, the assay for these antibodies has involved a variety of DNA sources and systems to detect DNA-anti-DNA interactions. The influence of these variations on antibody detection has complicated assay standardization. As an antigen, DNA has unique features since it is a highly charged polymer that has structural heterogeneity. This heterogeneity can affect antigenicity which can vary on the basis of DNA origin, size, conformation and mobility. In addition, as a polymer, DNA can promote patterns of antibody binding based on monogamous or bivalent interaction which require an extended polynucleotide structure. Understanding the nature of DNA as an antigen can facilitate interpretation of serological tests and underpin efforts at better standardization.

  14. Working towards a consensus for antibody validation

    PubMed Central

    Reiss, Peter D.; Min, Danxi; Leung, Mei Y.

    2014-01-01

    Commercial research antibodies are the most commonly used product in the life science tools market, and their applications represent a significant investment of time and resources for researchers. Frequently however, the quality of antibodies does not meet the expectations of consumers, causing loss of valuable time and money. This can delay research efforts and scientific discovery, or even lead to false, irreproducible results to be published in the scientific literature. This raises the question of whether there should be universal standards for validating antibodies.   During the 1 st International Antibody Validation Forum, hosted by St John’s Laboratory Ltd on October 15 th 2014 at Queen Mary University of London, scientists from academia and industry presented data highlighting quality issues arising from lack of antibody validation. While the forum identified significant current problems in the antibody market, it also discussed future opportunities for improved quality and transparency by encouraging data disclosure and data sharing. This article highlights the key issues and conclusions reached at the forum. PMID:25580232

  15. Antibody-mediated resistance against plant pathogens.

    PubMed

    Safarnejad, Mohammad Reza; Jouzani, Gholamreza Salehi; Tabatabaei, Meisam; Tabatabaie, Meisam; Twyman, Richard M; Schillberg, Stefan

    2011-01-01

    Plant diseases have a significant impact on the yield and quality of crops. Many strategies have been developed to combat plant diseases, including the transfer of resistance genes to crops by conventional breeding. However, resistance genes can only be introgressed from sexually-compatible species, so breeders need alternative measures to introduce resistance traits from more distant sources. In this context, genetic engineering provides an opportunity to exploit diverse and novel forms of resistance, e.g. the use of recombinant antibodies targeting plant pathogens. Native antibodies, as a part of the vertebrate adaptive immune system, can bind to foreign antigens and eliminate them from the body. The ectopic expression of antibodies in plants can also interfere with pathogen activity to confer disease resistance. With sufficient knowledge of the pathogen life cycle, it is possible to counter any disease by designing expression constructs so that pathogen-specific antibodies accumulate at high levels in appropriate sub-cellular compartments. Although first developed to tackle plant viruses and still used predominantly for this purpose, antibodies have been targeted against a diverse range of pathogens as well as proteins involved in plant-pathogen interactions. Here we comprehensively review the development and implementation of antibody-mediated disease resistance in plants.

  16. Modern affinity reagents: Recombinant antibodies and aptamers.

    PubMed

    Groff, Katherine; Brown, Jeffrey; Clippinger, Amy J

    2015-12-01

    Affinity reagents are essential tools in both basic and applied research; however, there is a growing concern about the reproducibility of animal-derived monoclonal antibodies. The need for higher quality affinity reagents has prompted the development of methods that provide scientific, economic, and time-saving advantages and do not require the use of animals. This review describes two types of affinity reagents, recombinant antibodies and aptamers, which are non-animal technologies that can replace the use of animal-derived monoclonal antibodies. Recombinant antibodies are protein-based reagents, while aptamers are nucleic-acid-based. In light of the scientific advantages of these technologies, this review also discusses ways to gain momentum in the use of modern affinity reagents, including an update to the 1999 National Academy of Sciences monoclonal antibody production report and federal incentives for recombinant antibody and aptamer efforts. In the long-term, these efforts have the potential to improve the overall quality and decrease the cost of scientific research. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. SPECIFIC FRACTIONATION OF HUMAN ANTIDEXTRAN ANTIBODIES

    PubMed Central

    Gelzer, Justus; Kabat, Elvin A.

    1964-01-01

    Human antidextran of one individual, absorbed specifically on sephadex, was fractionated into two populations of antibody molecules by successive elution with oligosaccharides of the isomaltose series of increasing size. The purified antibody fractions and some whole antidextran sera were found to fix complement with dextrans of molecular weight of 195,000 and above. It could be demonstrated by quantitative microcomplement fixation inhibition assays that the antibody eluted with isomaltotriose had a higher affinity for smaller oligosaccharides relative to isomaltohexaose, indicating a high content of antibody molecules with smaller combining sites, while with the second fraction, eluted with isomaltohexaose, the small haptens were very poor inhibitors and the larger oligosaccharides inhibited readily, presumably due to a higher proportion of molecules with larger combining site size. Assays of similarly prepared fractions, obtained from earlier bleedings of the same individual (1), with inhibition of complement fixation were in good agreement with those obtained by inhibition of precipitation. The two purified antidextran fractions were shown to differ with respect to their complement-fixing capacity. The fraction with molecules with smaller size-combining sites fixed only about half as much complement per unit antibody N as did the fraction containing largely molecules with larger combining sites suggesting that the strength of complement fixation is affected by the strength of the antigen-antibody interaction. PMID:14176295

  18. A mutagenesis study of a catalytic antibody

    SciTech Connect

    Jackson, D.Y.; Prudent, J.R.; Baldwin, E.P.; Schultz, P.G. )

    1991-01-01

    The authors have generated seven site-specific mutations in the genes encoding the variable region of the heavy chain domain (V{sub H}) of the phosphocholine-binding antibody S107.S107 is a member of a family of well-characterized highly homologous antibodies that bind phosphorylcholine mono- and diesters. Two of these antibodies, MOPC-167 and T15, have previously been shown to catalyze the hydrolysis of 4-nitrophenyl N-trimethylammonioethyl carbonate. Two conserved heavy-chain residues, Tyr-33 and Arg-52, were postulated to be involved in binding and hydrolysis of 4-nitrophenylcholine carbonate esters. To more precisely define the catalytic roles of these residues, three Arg-52 mutants (R52K, R52Q, R52C) and four Tyr-33 mutants (Y33H, Y33F, Y33E, Y33D) of antibody S107 were generated. The genes encoding the V{sub H} binding domain of S107 were inserted into plasmid pUC-fl, and in vitro mutagenesis was performed. These results not only demonstrate the importance of electrostatic interactions in catalysis by antibody S107 but also show that catalytic side chains can be introduced into antibodies to enhance their catalytic efficiency.

  19. Fetal arthrogryposis and maternal serum antibodies.

    PubMed

    Dalton, Paola; Clover, Linda; Wallerstein, Robert; Stewart, Helen; Genzel-Boroviczeny, Orsolya; Dean, Andrew; Vincent, Angela

    2006-08-01

    Arthrogryposis multiplex congenital (AMC) describes multiple joint contractures resulting from lack of movement in utero. Antibodies directed at the fetal isoform of the muscle acetylcholine receptor (AChR) have been reported in a small number of asymptomatic mothers of AMC babies. We examined sera from 179 mothers of AMC babies and 20 parous and non-parous controls to look for antibodies to AChR or undefined muscle or neuronal proteins. We found positive AChR antibodies in only three sera (1.5%) from asymptomatic AMC mothers. However, there was reactivity with muscle or with neuronal antigens in 33% of the sera, and reactivity to undefined neuronal antigens was more common in sera from mothers of AMC babies with CNS involvement (p=0.001) than those without. The offspring of mothers with AChR antibodies may benefit from treatment during pregnancy. Other maternal antibodies require further study, but these observations add to the emerging literature on maternal antibodies associated with developmental intrauterine disorders.

  20. Working towards a consensus for antibody validation.

    PubMed

    Reiss, Peter D; Min, Danxi; Leung, Mei Y

    2014-01-01

    Commercial research antibodies are the most commonly used product in the life science tools market, and their applications represent a significant investment of time and resources for researchers. Frequently however, the quality of antibodies does not meet the expectations of consumers, causing loss of valuable time and money. This can delay research efforts and scientific discovery, or even lead to false, irreproducible results to be published in the scientific literature. This raises the question of whether there should be universal standards for validating antibodies.   During the 1 (st) International Antibody Validation Forum, hosted by St John's Laboratory Ltd on October 15 (th) 2014 at Queen Mary University of London, scientists from academia and industry presented data highlighting quality issues arising from lack of antibody validation. While the forum identified significant current problems in the antibody market, it also discussed future opportunities for improved quality and transparency by encouraging data disclosure and data sharing. This article highlights the key issues and conclusions reached at the forum.

  1. Delivery of antibodies to the cytosol

    PubMed Central

    Marschall, Andrea LJ; Zhang, Congcong; Frenzel, André; Schirrmann, Thomas; Hust, Michael; Perez, Franck; Dübel, Stefan

    2014-01-01

    The use of antibodies to target their antigens in living cells is a powerful analytical tool for cell biology research. Not only can molecules be localized and visualized in living cells, but interference with cellular processes by antibodies may allow functional analysis down to the level of individual post-translational modifications and splice variants, which is not possible with genetic or RNA-based methods. To utilize the vast resource of available antibodies, an efficient system to deliver them into the cytosol from the outside is needed. Numerous strategies have been proposed, but the most robust and widely applicable procedure still remains to be identified, since a quantitative ranking of the efficiencies has not yet been done. To achieve this, we developed a novel efficiency evaluation method for antibody delivery based on a fusion protein consisting of a human IgG1 Fc and the recombination enzyme Cre (Fc-Cre). Applied to suitable GFP reporter cells, it allows the important distinction between proteins trapped in endosomes and those delivered to the cytosol. Further, it ensures viability of positive cells and is unsusceptible to fixation artifacts and misinterpretation of cellular localization in microscopy and flow cytometry. Very low cytoplasmic delivery efficiencies were found for various profection reagents and membrane penetrating peptides, leaving electroporation as the only practically useful delivery method for antibodies. This was further verified by the successful application of this method to bind antibodies to cytosolic components in living cells. PMID:24848507

  2. Secretory antibody following oral influenza immunization.

    PubMed

    Waldman, R H; Stone, J; Bergmann, K C; Khakoo, R; Lazzell, V; Jacknowitz, A; Waldman, E R; Howard, S

    1986-12-01

    Secretory IgA antibody may be important in protection against respiratory viral infections, and the concept of a common mucosal immune system offers the theoretical basis for the convenient stimulation of this antibody. Therefore, the oral route was compared with intramuscular injection in a double-blind, placebo-controlled study in young healthy volunteers. A killed influenza vaccine, given in enteric-coated capsules (total of 98 ug hemagglutinin of A/Bangkok) led to significant salivary and nasal IgA antibody rises in a 4-week period. The preimmunization titers in secretions were inversely correlated with the antibody rise after immunization. The orally administered vaccine was associated with no more side effects than placebo, in contradistinction to reactions following the intramuscular route. The latter route also was without significant effect in regard to a stimulation of secretory antibodies. The observed simultaneous induction of antibodies in saliva and nasal secretions following oral administration of killed vaccine gives further evidence of a common mucosal immune system and its possible clinical use.

  3. Monoclonal antibodies to leukotoxin of Actinobacillus actinomycetemcomitans.

    PubMed Central

    DiRienzo, J M; Tsai, C C; Shenker, B J; Taichman, N S; Lally, E T

    1985-01-01

    Hybridoma cell lines which produce monoclonal antibodies to a leukotoxin from Actinobacillus actinomycetemcomitans were prepared. The monoclonal antibodies were selected for their ability to neutralize the cytotoxic activity of the leukotoxin and recognize the toxin on nitrocellulose blots. The antibodies belonged to either the immunoglobulin G1 (IgG1) or IgG2 subclass and differed in their ability to bind to the leukotoxin on nitrocellulose blots. However, only slight differences in neutralization titers were observed. Use of the monoclonal antibodies revealed that polymyxin B-extracted or osmotic shock-released leukotoxin could be separated into several high-molecular-weight polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot analysis with the monoclonal antibodies also demonstrated that the leukotoxin was present in eight oral strains of A. actinomycetemcomitans that had been previously classified by a biological assay as leukotoxic. The availability of these monoclonal antibodies should facilitate and expand studies concerning the role of the leukotoxin in the pathogenicity of A. actinomycetemcomitans. Images PMID:3965404

  4. Discovery of functional antibodies targeting ion channels.

    PubMed

    Wilkinson, Trevor C I; Gardener, Matthew J; Williams, Wendy A

    2015-04-01

    Ion channels play critical roles in physiology and disease by modulation of cellular functions such as electrical excitability, secretion, cell migration, and gene transcription. Ion channels represent an important target class for drug discovery that has been largely addressed, to date, using small-molecule approaches. A significant opportunity exists to target these channels with antibodies and alternative formats of biologics. Antibodies display high specificity and affinity for their target antigen, and they have the potential to target ion channels very selectively. Nevertheless, isolating antibodies to this target class is challenging due to the difficulties in expression and purification of ion channels in a format suitable for antibody drug discovery in addition to the complexity of screening for function. In this article, we will review the current state of ion channel biologics discovery and the progress that has been made. We will also highlight the challenges in isolating functional antibodies to these targets and how these challenges may be addressed. Finally, we also illustrate successful approaches to isolating functional monoclonal antibodies targeting ion channels by way of a number of case studies drawn from recent publications. © 2014 Society for Laboratory Automation and Screening.

  5. Boosting antibody developability through rational sequence optimization

    PubMed Central

    Seeliger, Daniel; Schulz, Patrick; Litzenburger, Tobias; Spitz, Julia; Hoerer, Stefan; Blech, Michaela; Enenkel, Barbara; Studts, Joey M; Garidel, Patrick; Karow, Anne R

    2015-01-01

    The application of monoclonal antibodies as commercial therapeutics poses substantial demands on stability and properties of an antibody. Therapeutic molecules that exhibit favorable properties increase the success rate in development. However, it is not yet fully understood how the protein sequences of an antibody translates into favorable in vitro molecule properties. In this work, computational design strategies based on heuristic sequence analysis were used to systematically modify an antibody that exhibited a tendency to precipitation in vitro. The resulting series of closely related antibodies showed improved stability as assessed by biophysical methods and long-term stability experiments. As a notable observation, expression levels also improved in comparison with the wild-type candidate. The methods employed to optimize the protein sequences, as well as the biophysical data used to determine the effect on stability under conditions commonly used in the formulation of therapeutic proteins, are described. Together, the experimental and computational data led to consistent conclusions regarding the effect of the introduced mutations. Our approach exemplifies how computational methods can be used to guide antibody optimization for increased stability. PMID:25759214

  6. Construction of human antibody gene libraries and selection of antibodies by phage display.

    PubMed

    Frenzel, André; Kügler, Jonas; Wilke, Sonja; Schirrmann, Thomas; Hust, Michael

    2014-01-01

    Antibody phage display is the most commonly used in vitro selection technology and has yielded thousands of useful antibodies for research, diagnostics, and therapy.The prerequisite for successful generation and development of human recombinant antibodies using phage display is the construction of a high-quality antibody gene library. Here, we describe the methods for the construction of human immune and naive scFv gene libraries.The success also depends on the panning strategy for the selection of binders from these libraries. In this article, we describe a panning strategy that is high-throughput compatible and allows parallel selection in microtiter plates.

  7. Competitive exclusion by autologous antibodies can prevent broad HIV-1 antibodies from arising

    DOE PAGES

    Luo, Shishi; Perelson, Alan S.

    2015-08-31

    The past decade has seen the discovery of numerous broad and potent monoclonal antibodies against HIV type 1 (HIV-1). Eliciting these antibodies via vaccination appears to be remarkably difficult, not least because they arise late in infection and are highly mutated relative to germline antibody sequences. Here, using a computational model, we show that broad antibodies could in fact emerge earlier and be less mutated, but that they may be prevented from doing so as a result of competitive exclusion by the autologous antibody response. We further find that this competitive exclusion is weaker in infections founded by multiple distinctmore » strains, with broadly neutralizing antibodies emerging earlier than in infections founded by a single strain. Our computational model simulates coevolving multitype virus and antibody populations. Broadly neutralizing antibodies may therefore be easier for the adaptive immune system to generate than previously thought. As a result, if less mutated broad antibodies exist, it may be possible to elicit them with a vaccine containing a mixture of diverse virus strains.« less

  8. Competitive exclusion by autologous antibodies can prevent broad HIV-1 antibodies from arising

    SciTech Connect

    Luo, Shishi; Perelson, Alan S.

    2015-08-31

    The past decade has seen the discovery of numerous broad and potent monoclonal antibodies against HIV type 1 (HIV-1). Eliciting these antibodies via vaccination appears to be remarkably difficult, not least because they arise late in infection and are highly mutated relative to germline antibody sequences. Here, using a computational model, we show that broad antibodies could in fact emerge earlier and be less mutated, but that they may be prevented from doing so as a result of competitive exclusion by the autologous antibody response. We further find that this competitive exclusion is weaker in infections founded by multiple distinct strains, with broadly neutralizing antibodies emerging earlier than in infections founded by a single strain. Our computational model simulates coevolving multitype virus and antibody populations. Broadly neutralizing antibodies may therefore be easier for the adaptive immune system to generate than previously thought. As a result, if less mutated broad antibodies exist, it may be possible to elicit them with a vaccine containing a mixture of diverse virus strains.

  9. Nanopore-based kinetics analysis of individual antibody-channel and antibody-antigen interactions

    PubMed Central

    Winters-Hilt, Stephen; Morales, Eric; Amin, Iftekhar; Stoyanov, Alexander

    2007-01-01

    Background The UNO/RIC Nanopore Detector provides a new way to study the binding and conformational changes of individual antibodies. Many critical questions regarding antibody function are still unresolved, questions that can be approached in a new way with the nanopore detector. Results We present evidence that different forms of channel blockade can be associated with the same antibody, we associate these different blockades with different orientations of "capture" of an antibody in the detector's nanometer-scale channel. We directly detect the presence of antibodies via reductions in channel current. Changes to blockade patterns upon addition of antigen suggest indirect detection of antibody/antigen binding. Similarly, DNA-hairpin anchored antibodies have been studied, where the DNA linkage is to the carboxy-terminus at the base of the antibody's Fc region, with significantly fewer types of (lengthy) capture blockades than was observed for free (un-bound) IgG antibody. The introduction of chaotropic agents and its effects on protein-protein interactions have also been observed. Conclusion Nanopore-based approaches may eventually provide a direct analysis of the complex conformational "negotiations" that occur upon binding between proteins. PMID:18047720

  10. Competitive exclusion by autologous antibodies can prevent broad HIV-1 antibodies from arising.

    PubMed

    Luo, Shishi; Perelson, Alan S

    2015-09-15

    The past decade has seen the discovery of numerous broad and potent monoclonal antibodies against HIV type 1 (HIV-1). Eliciting these antibodies via vaccination appears to be remarkably difficult, not least because they arise late in infection and are highly mutated relative to germline antibody sequences. Here, using a computational model, we show that broad antibodies could in fact emerge earlier and be less mutated, but that they may be prevented from doing so as a result of competitive exclusion by the autologous antibody response. We further find that this competitive exclusion is weaker in infections founded by multiple distinct strains, with broadly neutralizing antibodies emerging earlier than in infections founded by a single strain. Our computational model simulates coevolving multitype virus and antibody populations. Broadly neutralizing antibodies may therefore be easier for the adaptive immune system to generate than previously thought. If less mutated broad antibodies exist, it may be possible to elicit them with a vaccine containing a mixture of diverse virus strains.

  11. Avian Diagnostic and Therapeutic Antibodies to Viral Emerging Pathogens

    SciTech Connect

    David Bradley

    2011-03-31

    During the current period the following key objectives were achieved: demonstration of high titer antibody production by geese following immunization with inactived H1N1 virus; completion of the epitope mapping of West Nile Virus-specific goose antibodies and initiation of epitope mapping of H1N1 flu-specific goose antibodies; advancement in scalable purification of goose antibodies.

  12. Antiphospholipid Antibody Syndrome Presenting with Unilateral Adrenal Hemmorhage.

    PubMed

    Ullah, Kifayat; Butt, Ghias; Neopane, Sippy; Arshi, Shahana

    2016-06-01

    The antiphospholipid antibody syndrome presents with vascular thrombosis which involve both arterial and venous systems. The clinical presentation of antiphospholipid antibody syndrome includes obstetric complications leading to recurrent abortions, presence of circulating antibodies against phospholipids, and multi-organ thromboembolisms. We report a case of a patient who presented with unilateral adrenal hemorrhage and subsequently found to have antiphospholipid antibody syndrome and lupus nephritis.

  13. Antibody Scientific Committee | Office of Cancer Clinical Proteomics Research

    Cancer.gov

    The Antibody Scientific Committee provides scientific insight and guidance to the NCI's Antibody Characterization Program. Specifically, the members of this committee evaluate request from the external scientific community for development and characterization of antibodies by the program. The members of the Antibody Scientific Committee include:

  14. 21 CFR 866.5100 - Antinuclear antibody immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Antinuclear antibody immunological test system....5100 Antinuclear antibody immunological test system. (a) Identification. An antinuclear antibody... the autoimmune antibodies in serum, other body fluids, and tissues that react with cellular nuclear...

  15. 21 CFR 866.5100 - Antinuclear antibody immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Antinuclear antibody immunological test system....5100 Antinuclear antibody immunological test system. (a) Identification. An antinuclear antibody... the autoimmune antibodies in serum, other body fluids, and tissues that react with cellular nuclear...

  16. [Advances in the study of natural small molecular antibody].

    PubMed

    Zhu, Lei; Zhang, Da-peng

    2012-10-01

    Small molecule antibodies are naturally existed and well functioned but not structurally related to the conventional antibodies. They are only composed of heavy protein chains or light chains, much smaller than common antibody. The first small molecule antibody, called Nanobody was engineered from heavy-chain antibodies found in camelids. Cartilaginous fishes also have heavy-chain antibodies (IgNAR, "immunoglobulin new antigen receptor"), from which single-domain antibodies called Vnar fragments can be obtained. In addition, free light chain (FLC) antibodies in human bodies are being developed as therapeutic and diagnostic agents. Comparing to intact antibodies, common advantages of small molecule antibodies are with better solubility, tissue penetration, stability towards heat and enzymes, and comparatively low production costs. This article reviews the structural characteristics and mechanism of action of the Nanobody, IgNAR and FLC.

  17. Coming-of-Age of Antibodies in Cancer Therapeutics.

    PubMed

    Ayyar, B Vijayalakshmi; Arora, Sushrut; O'Kennedy, Richard

    2016-12-01

    Antibody-based therapies have garnered considerable success in recent years. This is due to the availability of strategies to successfully engineer antibodies into humanized forms, better understanding of the biological processes involved in cancer development, the availability of novel recombinant antibody formats, better antibody selection platforms, and improved antibody conjugation methodologies. Such achievements have led to an explosion in the generation of antibodies and antibody-associated constructs for the treatment of cancer and other diseases. In this review, we critically assess recent trends in the development and applications of bispecific antibodies (bsAbs), antibody-drug conjugates (ADCs), and immune checkpoint inhibitors (ICIs) as cancer therapeutics. We also highlight recent US FDA approvals and clinical trials of antibody-based cancer therapies.

  18. [Progress in monoclonal antibody-based immunotherapy for cancer treatment].

    PubMed

    Guo, Yajun

    2015-06-01

    More than 100 years ago, Paul Ehrlich first proposed the "magic bullets" concept in which antibody targeting disease related antigen can fight against human disease. Since then, with the development of hybridoma technology for monoclonal antibody production and cancer serum therapy, immunotherapy based monoclonal antibody bas been used in chinical practice to treat hematological and solid tumor. Up to now, more than 20 recombinant antibody drugs were approved for cancer treatment worldwide. In recent years, the next-generation antibody drug, including immune checkpoint antagonists, bi-specific antibody, and antibody drug conjugates have successfully cured various malignant tumor. This review recalled the history of monoclonal antibody as potent immunotherapy of cancer firstly, and focused on the next-generation antibody drug's mechanism of action, construction strategies, and the side effects in clinic. Lastly, the future trend of anti-tumor antibody drug was also discussed.

  19. Immunoglobulin VH determinants defined by monoclonal antibodies.

    PubMed

    Kubagawa, H; Mayumi, M; Kearney, J F; Cooper, M D

    1982-10-01

    Hybridoma clones secreting antibodies against common VH determinants were readily produced by fusion of cells from mice immunized with isolated V mu fragments of human immunoglobulins (Ig), but not with intact Ig molecules or isolated heavy chains. Four monoclonal antibodies to the V mu fragments of different IgM paraproteins were selected for analysis: MH-44 (mu kappa), GB-24 (mu kappa), NF-11 (gamma 1 kappa), and SA-44 (gamma 1 kappa). Each antibody reacted with the homologous V mu fragment, homologous mu chain, and normal gamma chains, but not with the intact IgM molecules, intact IgG, or isolated light chains, as determined by radioimmunoassay. The VH reaction spectra with a panel of myeloma heavy chains showed overlapping but distinctive patterns for the four antibodies. Each of the four monoclonal anti-VH antibodies appeared to react with a different "hidden" VH determinant that is not exposed on undenatured, intact Ig molecules and differs from conventional VH subgroup determinants. In immunofluorescence studies, the monoclonal anti-VH antibodies did not bind to surface Ig on viable B lymphocytes, but visibly stained subpopulations of fixed B lymphocytes, pre-B cells, and normal plasma cells. The mean frequencies of VH+ plasma cells were 30% (MH-44), 17% (GB-24), 13% (NF-11), and 3% (SA-44), and similar frequencies were obtained for the VH+ B cell subpopulations. While subpopulations of B cells could be identified at all stages in differentiation by immunofluorescence with the anti-VH antibodies, neither resting nor activated T cells expressed these VH determinants in detectable amounts.

  20. The significance of anti-streptokinase antibodies.

    PubMed Central

    Lynch, M; Pentecost, B L; Littler, W A; Stockley, R A

    1994-01-01

    Antibodies to streptokinase (SK) are widespread in the population, but reports of their effect on the action of SK are conflicting. Specific anti-SK IgG was purified from the sera of 10 patients, five with low titres of anti-SK IgG and five with high titres. The effect of increasing specific anti-SK IgG antibodies on the action of SK was evaluated in vitro using a fluorimetric assay for plasmin and by a fibrin plate lysis assay. The inhibition of SK by whole plasma from a further group of patients was also assessed by the fibrin plate assay. There was a positive correlation between the serum antibody concentration and the quantity of specific anti-SK eluted (r = 0.797; P < 0.005). The addition of specific anti-SK IgG caused a dose-related decrease in SK activity (fluorimetric assay r = -0.93; P = 0.02; fibrin plate assay r = -0.98; P < 0.001). The addition of patient plasma to the fibrin plate assay also resulted in decreased lysis, which was dependent upon antibody titre (r = -0.95; P < 0.0001). Significant in vitro reduction of the activity of SK by specific antibody was demonstrated, and this was similar with plasma containing comparable amounts of antibody. The findings suggest that treatment with SK would be unlikely to induce an effective thrombolytic state when antibody titres are high (such as those seen within 2 years of an initial dose of SK). PMID:8004811